Methods of eliciting an immune response

ABSTRACT

Provided are methods for eliciting an immune response, the methods comprising administering a vaccine to a subject. The vaccines for eliciting an immune response comprise RNA encoding an immunogen, which is delivered in a liposome, for the purposes of immunisation. The liposome includes lipids which have a pKa in the range of 5.0 to 7.6 and, preferably, a tertiary amine. These liposomes can have essentially neutral surface charge at physiological pH and are effective for immunisation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/808,080, filed Mar. 14, 2013, which is a U.S. National Phase ofInternational Application No. PCT/US2011/043105, filed Jul. 6, 2011 andpublished in English, which claims the benefit of U.S. ProvisionalApplication No. 61/361,830, filed Jul. 6, 2010, and U.S. ProvisionalApplication No. 61/378,837, filed Aug. 31, 2010. The complete contentsof the above-listed applications are hereby incorporated herein byreference for all purposes.

TECHNICAL FIELD

This invention is in the field of non-viral delivery of RNA forimmunisation.

BACKGROUND

The delivery of nucleic acids for immunising animals has been a goal forseveral years. Various approaches have been tested, including the use ofDNA or RNA, of viral or non-viral delivery vehicles (or even no deliveryvehicle, in a “naked” vaccine), of replicating or non-replicatingvectors, or of viral or non-viral vectors.

There remains a need for further and improved nucleic acid vaccines.

DISCLOSURE OF THE INVENTION

According to the invention, RNA encoding an immunogen is delivered in aliposome for the purposes of immunisation. The liposome includes lipidswhich have a pKa in the range of 5.0 to 7.6. Ideally the lipid with apKa in this range has a tertiary amine; such lipids behave differentlyfrom lipids such as DOTAP or DC-Chol, which have a quaternary aminegroup. At physiological pH amines with a pKa in the range of 5.0 to 7.6have neutral or reduced surface charge, whereas a lipid such as DOTAP isstrongly cationic. The inventors have found that liposomes formed fromquaternary amine lipids (e.g. DOTAP) are less suitable for delivery ofimmunogen-encoding RNA than liposomes formed from tertiary amine lipids(e.g. DLinDMA).

Thus the invention provides a liposome having a lipid bilayerencapsulating an aqueous core, wherein: (i) the lipid bilayer comprisesa lipid having a pKa in the range of 5.0 to 7.6, and preferably having atertiary amine; and (ii) the aqueous core includes a RNA which encodesan immunogen. These liposomes are suitable for in vivo delivery of theRNA to a vertebrate cell and so they are useful as components inpharmaceutical compositions for immunising subjects against variousdiseases.

The invention also provides a process for preparing a RNA-containingliposome, comprising steps of: (a) mixing RNA with a lipid at a pH whichis below the lipid's pKa but is above 4.5, to form a liposome in whichthe RNA is encapsulated; and (b) increasing the pH of the resultingliposome-containing mixture to be above the lipid's pKa.

The Liposome

The invention utilises liposomes in which immunogen-encoding RNA isencapsulated. Thus the RNA is (as in a natural virus) separated from anyexternal medium by the liposome's lipid bilayer, and encapsulation inthis way has been found to protect RNA from RNase digestion. Theliposomes can include some external RNA (e.g. on their surface), but atleast half of the RNA (and ideally all of it) is encapsulated in theliposome's core. Encapsulation within liposomes is distinct from, forinstance, the lipid/RNA complexes disclosed in reference 1.

Various amphiphilic lipids can form bilayers in an aqueous environmentto encapsulate a RNA-containing aqueous core as a liposome. These lipidscan have an anionic, cationic or zwitterionic hydrophilic head group.Liposomes of the invention comprise a lipid having a pKa in the range of5.0 to 7.6, and preferred lipids with a pKa in this range have atertiary amine. For example, they may comprise1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA; pKa 5.8) and/or1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA). Anothersuitable lipid having a tertiary amine is1,2-dioleyloxy-N,Ndimethyl-3-aminopropane (DODMA). See FIG. 3 &reference 2. Some of the amino acid lipids of reference 3 may also beused, as can certain of the amino lipids of reference 4. Further usefullipids with tertiary amines in their headgroups are disclosed inreference 5, the complete contents of which are incorporated herein byreference.

Liposomes of the invention can be formed from a single lipid or from amixture of lipids, provided that at least one of the lipids has a pKa inthe range of 5.0 to 7.6 (and, preferably, a tertiary amine). Within thispKa range, preferred lipids have a pKa of 5.5 to 6.7 e.g. between 5.6and 6.8, between 5.6 and 6.3, between 5.6 and 6.0, between 5.5 and 6.2,or between 5.7 and 5.9. The pKa is the pH at which 50% of the lipids arecharged, lying halfway between the point where the lipids are completelycharged and the point where the lipids are completely uncharged. It canbe measured in various ways, but is preferably measured using the methoddisclosed below in the section entitled “pKa measurement”. The pKatypically should be measured for the lipid alone rather than for thelipid in the context of a mixture which also includes other lipids (e.g.not as performed in reference 6, which looks at the pKa of a SNALPrather than of the individual lipids).

Where a liposome of the invention is formed from a mixture of lipids, itis preferred that the proportion of those lipids which have a pKa withinthe desired range should be between 20-80% of the total amount of lipidse.g. between 30-70%, or between 40-60%. For instance, useful liposomesare shown below in which 40% or 60% of the total lipid is a lipid with apKa in the desired range. The remainder can be made of e.g. cholesterol(e.g. 35-50% cholesterol) and/or DMG (optionally PEGylated) and/or DSPC.Such mixtures are used below. These % values are mole percentages.

A liposome may include an amphiphilic lipid whose hydrophilic portion isPEGylated (i.e. modified by covalent attachment of a polyethyleneglycol). This modification can increase stability and preventnon-specific adsorption of the liposomes. For instance, lipids can beconjugated to PEG using techniques such as those disclosed in references6 and 7. PEG provides the liposomes with a coat which can conferfavourable pharmacokinetic characteristics. The combination of efficientencapsulation of a RNA (particularly a self-replicating RNA), a cationiclipid having a pKa in the range 5.0-7.6, and a PEGylated surface, allowsfor efficient delivery to multiple cell types (including both immune andnon-immune cells), thereby eliciting a stronger and better immuneresponse than when using quaternary amines without PEGylation. Variouslengths of PEG can be used e.g. between 0.5-8 kDa.

Lipids used with the invention can be saturated or unsaturated. The useof at least one unsaturated lipid for preparing liposomes is preferred.FIG. 3 shows three useful unsaturated lipids. If an unsaturated lipidhas two tails, both tails can be unsaturated, or it can have onesaturated tail and one unsaturated tail.

A mixture of DSPC, DLinDMA, PEG-DMG and cholesterol is used in theexamples. An independent aspect of the invention is a liposomecomprising DSPC, DLinDMA, PEG-DMG & cholesterol. This liposomepreferably encapsulates RNA, such as a self-replicating RNA e.g.encoding an immunogen.

Liposomal particles are usually divided into three groups: multilamellarvesicles (MLV); small unilamellar vesicles (SUV); and large unilamellarvesicles (LUV). MLVs have multiple bilayers in each vesicle, formingseveral separate aqueous compartments. SUVs and LUVs have a singlebilayer encapsulating an aqueous core; SUVs typically have a diameter≤50 nm, and LUVs have a diameter >50 nm. Liposomal particles of theinvention are ideally LUVs with a diameter in the range of 50-220 nm.For a composition comprising a population of LUVs with differentdiameters: (i) at least 80% by number should have diameters in the rangeof 20-220 nm, (ii) the average diameter (Zav, by intensity) of thepopulation is ideally in the range of 40-200 nm, and/or (iii) thediameters should have a polydispersity index <0.2. The liposome/RNAcomplexes of reference 1 are expected to have a diameter in the range of600-800 nm and to have a high polydispersity. The liposome can besubstantially spherical.

Techniques for preparing suitable liposomes are well known in the arte.g. see references 8 to 10. One useful method is described in reference11 and involves mixing (i) an ethanolic solution of the lipids (ii) anaqueous solution of the nucleic acid and (iii) buffer, followed bymixing, equilibration, dilution and purification. Preferred liposomes ofthe invention are obtainable by this mixing process.

Mixing Process

As mentioned above, the invention provides a process for preparing aRNA-containing liposome, comprising steps of: (a) mixing RNA with alipid at a pH which is below the lipid's pKa but is above 4.5; then (b)increasing the pH to be above the lipid's pKa.

Thus a cationic lipid is positively charged during liposome formation instep (a), but the pH change thereafter means that the majority (or all)of the positively charged groups become neutral. This process isadvantageous for preparing liposomes of the invention, and by avoiding apH below 4.5 during step (a) the stability of the encapsulated RNA isimproved.

The pH in step (a) is above 4.5, and is ideally above 4.8. Using a pH inthe range of 5.0 to 6.0, or in the range of 5.0 to 5.5, can providesuitable liposomes.

The increased pH in step (b) is above the lipid's pKa. The pH is ideallyincreased to a pH less than 9, and preferably less than 8. Depending onthe lipid's pKa, the pH in step (b) may thus be increased to be withinthe range of 6 to 8 e.g. to pH 6.5±0.3. The pH increase of step (b) canbe achieved by transferring the liposomes into a suitable buffer e.g.into phosphate-buffered saline. The pH increase of step (b) is ideallyperformed after liposome formation has taken place.

RNA used in step (a) can be in aqueous solution, for mixing with anorganic solution of the lipid (e.g. an ethanolic solution, as inreference 11). The mixture can then be diluted to form liposomes, afterwhich the pH can be increased in step (b).

Lipid Compositions

A composition may comprise a biologically active compound, optionally incombination with another lipid component.

The other lipid component(s) may be one or more selected from the groupconsisting of cationic lipids, neutral lipids, helper lipids, stealthlipids and alkyl resorcinol based lipids.

The other lipid component(s) may, for example, be (a) neutral lipid(s).The neutral lipid(s) may, in one embodiment, be one or more selectedfrom any of a variety of neutral uncharged or zwitterionic lipids.Examples of neutral phospholipids include:dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine,phosphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC),phosphatidylcholine (PLPC), phosphatidylethanolamine (PE), eggphosphatidylcholine (EPC), dilauryloylphosphatidylcholine (DLPC),dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl phosphatidylcholine(PMPC), 1-palmitoyl-2-stearoyl phosphatidylcholine (PSPC),1-stearoyl-2-palmitoyl phosphatidylcholine (SPPC),1,2-distearoyl-sn-glycero-3-phosphocholine (DAPC),1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC),1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloeoylphosphatidylcholine (POPC), lysophosphatidylcholine,dilinoleoylphosphatidylcholine distearoylphophatidylethanolamine (DSPE),dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), palmitoyloeoyl phosphatidylethanolamine(POPE), lysophosphatidylethanolamine or a combination thereof. In apreferred embodiment, the neutral phospholipid is selected from thegroup consisting of distearoylphosphatidylcholine and dimyristoylphosphatidylethanolamine (DMPE).

The total amount of lipid in the composition being administered is, inone embodiment, from about 5 to about 30 mg lipid per mg biologicallyactive compound (e.g. RNA), in another embodiment from about 5 to about25 mg lipid per mg biologically active compound (e.g. RNA), in anotherembodiment from about 7 to about 25 mg lipid per mg biologically activecompound (e.g. RNA) and in a preferred embodiment from about 7 to about15 mg lipid per mg biologically active compound (e.g. RNA).

In one embodiment, the composition comprises a cationic lipid componentwhich forms from about 10% to about 80%, from about 20% to about 70% orfrom about 30% to about 60% of the total lipid present in thecomposition. These percentages are mole percentages relative to thetotal moles of lipid components in the final lipid particle.

In one embodiment, the composition comprises a neutral lipid componentwhich forms from about 0% to about 50%, from about 0% to about 30% orfrom about 10% to about 20% of the total lipid present in thecomposition. These percentages are mole percentages relative to thetotal moles of lipid components in the final lipid particle.

In one embodiment, the composition comprises a helper lipid componentwhich forms from about 5% to about 80%, from about 20% to about 70% orfrom about 30% to about 50% of the total lipid present in thecomposition. These percentages are mole percentages relative to thetotal moles of lipid components in the final lipid particle.

In one embodiment, the composition comprises a stealth lipid componentwhich forms from about 0% to about 10%, from about 1% to about 6%, orfrom about 2% to about 5% of the total lipid present in the composition.These percentages are mole percentages relative to the total moles oflipid components in the final lipid particle.

In one embodiment, the composition comprises a cationic lipid componentforming from about 30 to about 60% of the total lipid present in theformulation, a neutral lipid comprising forming from about 0 to about30% of the total lipid present in the formulation, a helper lipidforming from about 18 to about 46% of the total lipid present in theformulation and a stealth lipid forming from about 2 to about 4% of thetotal lipid present in the formulation. These percentages are molepercentages relative to the total moles of lipid components in the finallipid particle.

Helper Lipids

The term “helper lipid” as used herein is meant a lipid that enhancestransfection (e.g. transfection of the nanoparticle including thebiologically active compound) to some extent. The mechanism by which thehelper lipid enhances transfection may include, for example, enhancingparticle stability and/or enhancing membrane fusogenicity. Helper lipidsinclude steroids and alkyl resorcinols. Examples of helper lipids arecholesterol, estrogen, testosterone, progesterone, glucocortisone,cortisol, vitamin D, and/or retinoic acid. Steroids are thought tofunction as stabilizing lipids in that they help provide rigidity to theparticle.

The other lipid component(s) may, for example, be (a) neutral lipid(s).The neutral lipid(s) may, in one embodiment, be one or more selectedfrom any of a variety of neutral uncharged or zwitterionic lipids.Examples of neutral phospholipids include:dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine,phosphocholine (DOPC), dimyristoyl phosphatidylcholine (DMPC),phosphatidylcholine (PLPC), phosphatidylethanolamine (PE), eggphosphatidylcholine (EPC), dilauryloylphosphatidylcholine (DLPC),dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl phosphatidylcholine(PMPC), 1-palmitoyl-2-stearoyl phosphatidylcholine (PSPC),1-stearoyl-2-palmitoyl phosphatidylcholine (SPPC),1,2-distearoyl-sn-glycero-3-phosphocholine (DAPC),1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC),1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloeoylphosphatidylcholine (POPC), lysophosphatidylcholine,dilinoleoylphosphatidylcholine distearoylphophatidylethanolamine (DSPE),dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), palmitoyloeoyl phosphatidylethanolamine(POPE), lysophosphatidylethanolamine or a combination thereof. In apreferred embodiment, the neutral phospholipid is selected from thegroup consisting of distearoylphosphatidylcholine and DMPE.

The other lipid component(s) may, for example, be (a) anionic lipid(s),e.g. anionic lipids capable of producing a stable complex. Examples ofanionic lipids are phosphatidylglycerol, cardiolipin,diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanoloamine, N-succinyl phosphatidylethanolamine,N-glutaryl phosphatidylethanolamine and lysylphosphatidylglycerol.

Preparation of the Lipid Compositions

It is preferred that the compounds are administered in the form of lipidnanoparticles. Thus it is preferred that the compositions comprise lipidnanoparticles which comprise compounds and optionally one or more otherlipid components.

To achieve size reduction and/or to increase the homogeneity of size inthe particles, the skilled person may use the method steps set outbelow, experimenting with different combinations. Additionally, theskilled person could employ sonication, filtration or other sizingtechniques which are used in liposomal formulations.

The process for making a composition typically comprises providing anaqueous solution comprising a biologically active compound in a firstreservoir, providing a second reservoir comprising an organic solutionof the lipid(s) and then mixing the aqueous solution with the organiclipid solution. The first reservoir is optionally in fluid communicationwith the second reservoir. The mixing step is optionally followed by anincubation step, a filtration step, and a dilution and/or concentrationstep.

In one embodiment, the biologically active compound(s) and/or thelipid(s) is/are in a suitable buffer. In one embodiment, thebiologically active compound(s) is in an aqueous buffer such as acitrate buffer. In one embodiment, the lipid(s) is in an organic alcoholsuch as ethanol.

In one embodiment, the incubation step comprises allowing the solutionfrom the mixing step to stand in a vessel for about 0 to about 100 hours(preferably about 0 to about 24 hours) at about room temperature andoptionally protected from light.

In one embodiment, a dilution step follows the incubation step. Thedilution step may involve dilution with aqueous buffer (e.g. citratebuffer) for example using a pumping apparatus (e.g. a peristaltic pump).

In one embodiment, the filtration step is ultrafiltration. In oneembodiment, the ultrafiltration comprises concentration of the dilutedsolution followed by diafiltration, for example using a suitable pumpingsystem (e.g. pumping apparatus such as a peristaltic pump or equivalentthereof) in conjunction with a suitable ultrafiltration membrane (e.g.GE Hollow fiber cartridges or an equivalent thereof).

The process should result in the formation of lipid nanoparticles. Inone embodiment, the lipid nanoparticles comprise the biologically activecompound.

In one embodiment, the mixing step provides a clear single phase.

In one embodiment, after the mixing step, the organic solvent is removedto provide a suspension of particles, wherein the biologically activecompound is encapsulated by the lipid(s), e.g. in a lipid bilayer.

The selection of an organic solvent will typically involve considerationof solvent polarity and the ease with which the solvent can be removedat the later stages of particle formation.

The organic solvent, which is also used as a solubilizing agent, ispreferably in an amount sufficient to provide a clear single phasemixture of biologically active compounds and lipids.

The organic solvent may be selected from one or more (e.g. two) ofchloroform, dichloromethane, diethylether, cyclohexane, cyclopentane,benzene, toluene, methanol, and other aliphatic alcohols (e.g. C₁ to C₈)such as ethanol, propanol, isopropanol, butanol, tert-butanol,iso-butanol, pentanol and hexanol.

The mixing step can take place by any number of methods, for example bymechanical means such as a vortex mixer.

The methods used to remove the organic solvent will typically involvediafiltration or evaporation at reduced pressures or blowing a stream ofinert gas (e.g. nitrogen or argon) across the mixture.

In other embodiments, the method further comprises adding nonlipidpolycations which are useful to effect the transformation of cells usingthe present compositions. Examples of suitable non lipid polycationsinclude, but are limited to, hexadimethrine bromide (sold under thebrand name POLYBRENE®, from Aldrich Chemical Co., Milwaukee, Wis., USA)or other salts of hexadimethrine. Other suitable polycations include,for example, salts of poly-L-ornithine, poly-L-arginine, poly-L-lysine,poly-D-lysine, polyallylamine and polyethyleneimine.

In certain embodiments, the formation of the lipid nanoparticles can becarried out either in a mono-phase system (e.g. a Bligh and Dyermonophase or similar mixture of aqueous and organic solvents) or in atwo-phase system with suitable mixing.

The lipid nanoparticle may be formed in a mono- or a bi- phase system.In a mono-phase system, the cationic lipid(s) and biologically activecompound are each dissolved in a volume of the mono-phase mixture.Combining the two solutions provides a single mixture in which thecomplexes form. In a bi-phase system, the cationic lipids bind to thebiologically active compound (which is present in the aqueous phase),and “pull” it into the organic phase.

In one embodiment, the lipid nanoparticles are prepared by a methodwhich comprises: (a) contacting the biologically active compound with asolution comprising noncationic lipids and a detergent to form acompound-lipid mixture; (b) contacting cationic lipids with thecompound-lipid mixture to neutralize a portion of the negative charge ofthe biologically active compound and form a charge-neutralized mixtureof biologically active compound and lipids; and (c) removing thedetergent from the charge-neutralized mixture.

In one group of embodiments, the solution of neutral lipids anddetergent is an aqueous solution. Contacting the biologically activecompound with the solution of neutral lipids and detergent is typicallyaccomplished by mixing together a first solution of the biologicallyactive compound and a second solution of the lipids and detergent.Preferably, the biologically active compound solution is also adetergent solution. The amount of neutral lipid which is used in thepresent method is typically determined based on the amount of cationiclipid used, and is typically of from about 0.2 to 5 times the amount ofcationic lipid, preferably from about 0.5 to about 2 times the amount ofcationic lipid used.

The biologically active compound-lipid mixture thus formed is contactedwith cationic lipids to neutralize a portion of the negative chargewhich is associated with the molecule of interest (or other polyanionicmaterials) present. The amount of cationic lipids used is typically theamount sufficient to neutralize at least 50% of the negative charge ofthe biologically active compound. Preferably, the negative charge willbe at least 70% neutralized, more preferably at least 90% neutralized.

The methods used to remove the detergent typically involve dialysis.When organic solvents are present, removal is typically accomplished bydiafiltration or evaporation at reduced pressures or by blowing a streamof inert gas (e.g. nitrogen or argon) across the mixture.

There is herein disclosed an apparatus for making a composition. Theapparatus typically includes a first reservoir for holding an aqueoussolution comprising a biologically active compound and a secondreservoir for holding an organic lipid solution. The apparatus alsotypically includes a pump mechanism configured to pump the aqueous andthe organic lipid solutions into a mixing region or mixing chamber atsubstantially equal flow rates. In one embodiment, the mixing region ormixing chamber comprises a T coupling or equivalent thereof, whichallows the aqueous and organic fluid streams to combine as input intothe T connector and the resulting combined aqueous and organic solutionsto exit out of the T connector into a collection reservoir or equivalentthereof.

The RNA

The invention is useful for in vivo delivery of RNA which encodes animmunogen. The RNA is translated by non-immune cells at the deliverysite, leading to expression of the immunogen, and it also causes immunecells to secrete type I interferons and/or pro-inflammatory cytokineswhich provide a local adjuvant effect. The non-immune cells may alsosecrete type I interferons and/or pro-inflammatory cytokines in responseto the RNA.

The RNA is +-stranded, and so it can be translated by the non-immunecells without needing any intervening replication steps such as reversetranscription. It can also bind to TLR7 receptors expressed by immunecells, thereby initiating an adjuvant effect.

Preferred +-stranded RNAs are self-replicating. A self-replicating RNAmolecule (replicon) can, when delivered to a vertebrate cell evenwithout any proteins, lead to the production of multiple daughter RNAsby transcription from itself (via an antisense copy which it generatesfrom itself). A self-replicating RNA molecule is thus typically a+-strand molecule which can be directly translated after delivery to acell, and this translation provides a RNA-dependent RNA polymerase whichthen produces both antisense and sense transcripts from the deliveredRNA. Thus the delivered RNA leads to the production of multiple daughterRNAs. These daughter RNAs, as well as collinear subgenomic transcripts,may be translated themselves to provide in situ expression of an encodedimmunogen, or may be transcribed to provide further transcripts with thesame sense as the delivered RNA which are translated to provide in situexpression of the immunogen. The overall results of this sequence oftranscriptions is a huge amplification in the number of the introducedreplicon RNAs and so the encoded immunogen becomes a major polypeptideproduct of the cells.

As shown below, a self-replicating activity is not required for a RNA toprovide an adjuvant effect, although it can enhance post-transfectionsecretion of cytokines. The self-replicating activity is particularlyuseful for achieving high level expression of the immunogen bynon-immune cells. It can also enhance apoptosis of the non-immune cells.

One suitable system for achieving self-replication is to use analphavirus-based RNA replicon. These +-stranded replicons are translatedafter delivery to a cell to give of a replicase (orreplicase-transcriptase). The replicase is translated as a polyproteinwhich auto-cleaves to provide a replication complex which createsgenomic −-strand copies of the +-strand delivered RNA. These −-strandtranscripts can themselves be transcribed to give further copies of the+-stranded parent RNA and also to give a subgenomic transcript whichencodes the immunogen. Translation of the subgenomic transcript thusleads to in situ expression of the immunogen by the infected cell.Suitable alphavirus replicons can use a replicase from a sindbis virus,a semliki forest virus, an eastern equine encephalitis virus, avenezuelan equine encephalitis virus, etc. Mutant or wild-type virussequences can be used e.g. the attenuated TC83 mutant of VEEV has beenused in replicons, as disclosed in reference 12.

A preferred self-replicating RNA molecule thus encodes (i) aRNA-dependent RNA polymerase which can transcribe RNA from theself-replicating RNA molecule and (ii) an immunogen. The polymerase canbe an alphavirus replicase e.g. comprising one or more of alphavirusproteins nsP1, nsP2, nsP3 and nsP4.

Whereas natural alphavirus genomes encode structural virion proteins inaddition to the non-structural replicase polyprotein, it is preferredthat a self-replicating RNA molecule of the invention does not encodealphavirus structural proteins. Thus a preferred self-replicating RNAcan lead to the production of genomic RNA copies of itself in a cell,but not to the production of RNA-containing virions. The inability toproduce these virions means that, unlike a wild-type alphavirus, theself-replicating RNA molecule cannot perpetuate itself in infectiousform. The alphavirus structural proteins which are necessary forperpetuation in wild-type viruses are absent from self-replicating RNAsof the invention and their place is taken by gene(s) encoding theimmunogen of interest, such that the subgenomic transcript encodes theimmunogen rather than the structural alphavirus virion proteins.

Thus a self-replicating RNA molecule useful with the invention may havetwo open reading frames. The first (5′) open reading frame encodes areplicase; the second (3′) open reading frame encodes an immunogen. Insome embodiments the RNA may have additional (e.g. downstream) openreading frames e.g. to encode further immunogens (see below) or toencode accessory polypeptides.

A self-replicating RNA molecule can have a 5′ sequence which iscompatible with the encoded replicase.

Self-replicating RNA molecules can have various lengths but they aretypically 5000-25000 nucleotides long e.g. 8000-15000 nucleotides, or9000-12000 nucleotides. Thus the RNA is longer than seen in siRNAdelivery.

A RNA molecule useful with the invention may have a 5′ cap (e.g. a7-methylguanosine). This cap can enhance in vivo translation of the RNA.

The 5′ nucleotide of a RNA molecule useful with the invention may have a5′ triphosphate group. In a capped RNA this may be linked to a7-methylguanosine via a 5′-to-5′ bridge. A 5′ triphosphate can enhanceRIG-I binding and thus promote adjuvant effects.

A RNA molecule may have a 3′ poly-A tail. It may also include a poly-Apolymerase recognition sequence (e.g. AAUAAA) near its 3′ end.

A RNA molecule useful with the invention will typically besingle-stranded. Single-stranded RNAs can generally initiate an adjuvanteffect by binding to TLR7, TLR8, RNA helicases and/or PKR. RNA deliveredin double-stranded form (dsRNA) can bind to TLR3, and this receptor canalso be triggered by dsRNA which is formed either during replication ofa single-stranded RNA or within the secondary structure of asingle-stranded RNA.

A RNA molecule useful with the invention can conveniently be prepared byin vitro transcription (IVT). IVT can use a (cDNA) template created andpropagated in plasmid form in bacteria, or created synthetically (forexample by gene synthesis and/or polymerase chain-reaction (PCR)engineering methods). For instance, a DNA-dependent RNA polymerase (suchas the bacteriophage T7, T3 or SP6 RNA polymerases) can be used totranscribe the RNA from a DNA template. Appropriate capping and poly-Aaddition reactions can be used as required (although the replicon'spoly-A is usually encoded within the DNA template). These RNApolymerases can have stringent requirements for the transcribed 5′nucleotide(s) and in some embodiments these requirements must be matchedwith the requirements of the encoded replicase, to ensure that theIVT-transcribed RNA can function efficiently as a substrate for itsself-encoded replicase.

As discussed in reference 13, the self-replicating RNA can include (inaddition to any 5′ cap structure) one or more nucleotides having amodified nucleobase. Thus the RNA can comprise m5C (5-methylcytidine),m5U (5-methyluridine), m6A (N6-methyladenosine), s2U (2-thiouridine), Um(2′-O-methyluridine), m1A (1-methyladenosine); m2A (2-methyladenosine);Am (2′-O-methyladenosine); ms2m6A (2-methylthio-N6-methyladenosine); i6A(N6-isopentenyladenosine); ms2i6A (2-methylthio-N6isopentenyladenosine);io6A (N6-(cis-hydroxyisopentenyl)adenosine); ms 2io6A(2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine); g6A(N6-glycinylcarbamoyladenosine); t6A (N6-threonyl carbamoyladenosine);ms2t6A (2-methylthio-N6-threonyl carbamoyladenosine); m6t6A(N6-methyl-N6-threonylcarbamoyladenosine);hn6A(N6.-hydroxynorvalylcarbamoyl adenosine); ms2hn6A(2-methylthio-N6-hydroxynorvalyl carbamoyladenosine); Ar(p)(2′-O-ribosyladenosine (phosphate)); I (inosine); m11 (1-methylinosine);m′Im (1,2′-O-dimethylinosine); m3C (3-methylcytidine); Cm(2T-O-methylcytidine); s2C (2-thiocytidine); ac4C (N4-acetylcytidine);f5C (5-fonnylcytidine); m5Cm (5,2-O-dimethylcytidine); ac4Cm(N4acetyl2TOmethylcytidine); k2C (lysidine); m1G (1-methylguanosine);m2G (N2-methylguanosine); m7G (7-methylguanosine); Gm(2′-O-methylguanosine); m22G (N2,N2-dimethylguanosine); m2Gm(N2,2′-O-dimethylguanosine); m22Gm (N2,N2,2′-O-trimethylguanosine);Gr(p) (2′-O-ribosylguanosine (phosphate)); yW (wybutosine); o2yW(peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodifiedhydroxywybutosine); imG (wyosine); mimG (methylguanosine); Q(queuosine); oQ (epoxyqueuosine); galQ (galtactosyl-queuosine); manQ(mannosyl-queuosine); preQo (7-cyano-7-deazaguanosine); preQi(7-aminomethyl-7-deazaguanosine); G (archaeosine); D (dihydrouridine);m5Um (5,2′-O-dimethyluridine); s4U (4-thiouridine); m5s2U(5-methyl-2-thiouridine); ouridine); s2Um (2-thio-2′-O-methyluridine);acp3U (3-(3-amino-3-carboxypropyl)uridine); ho5U (5-hydroxyuridine);mo5U (5-methoxyuridine); cmo5U (uridine 5-oxyacetic acid); mcmo5U(uridine 5-oxyacetic acid methyl ester); chm5U(5-(carboxyhydroxymethyl)uridine)); mchm5U(5-(carboxyhydroxymethyl)uridine methyl ester); mcm5U (5-methoxycarbonylmethyluridine); mcm5Um (S-methoxy carb onylmethyl-2-O-methyl uri dine);mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine); nm5s2U(5-aminomethyl-2-thiouridine); mnm5U (5-methylaminomethyluridine);mnm5s2U (5-methylaminomethyl-2-thiouridine); mnm5se2U(5-methylaminomethyl-2-selenouridine); ncm5U (5-carbamoylmethyluridine); ncm5Um (5-carbamoylmethyl-2′-O-methyluridine); cmnm5U (5-carboxymethylaminomethyluridine); cnmm5Um(5-carboxymethylaminomethyl-2-L-Omethyluridine); cmnm5s2U(5-carboxymethylaminomethyl-2-thiouridine); m62A(N6,N6-dimethyladenosine); Tm (2′-O-methylinosine); m4C(N4-methylcytidine); m4Cm (N4,2-O-dimethylcytidine); hm5C(5-hydroxymethylcytidine); m3U (3-methyluridine); cm5U(5-carboxymethyluridine); m6Am (N6,T-O-dimethyladenosine); rn62Am(N6,N6,O-2-trimethyladenosine); m2′7G (N2,7-dimethylguanosine); m2′2′7G(N2,N2,7-trimethylguanosine); m3Um (3,2T-O-dimethyluridine); m5D(5-methyldihy drouri dine); f5 Cm (5-formyl-2′ -O-methylcytidine); m1Gm(1,2′-O-dimethylguanosine); m′Am (1,2-O-dimethyl adenosine)irinomethyluridine); tm5s2U (S-taurinomethyl-2-thiouridine)); imG-14(4-demethyl guanosine); imG2 (isoguanosine); or ac6A(N6-acetyladenosine), hypoxanthine, inosine, 8-oxo-adenine,7-substituted derivatives thereof, dihydrouracil, pseudouracil,2-thiouracil, 4-thiouracil, 5-aminouracil, 5-(C1-C6)-alkyluracil,5-methyluracil, 5-(C2-C6)-alkenyluracil, 5-(C2-C6)-alkynyluracil,5-(hydroxymethyOuracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil,5-hydroxycytosine,

5-(C1 -C6)-alkylcytosine, 5-methylcytosine, 5-(C2-C6)-alkenylcytosine,5-(C2-C6)-alkynylcytosine, 5-chlorocytosine, 5-fluorocytosine,5-bromocytosine, N2-dimethylguanine, 7-deazaguanine, 8-azaguanine,7-deaza-7-substituted guanine, 7-deaza-7-(C2-C6)alkynylguanine,7-deaza-8-substituted guanine, 8-hydroxyguanine, 6-thioguanine,8-oxoguanine, 2-aminopurine, 2-amino-6-chloropurine, 2,4-diaminopurine,2,6-diaminopurine, 8-azapurine, substituted 7-deazapurine,7-deaza-7-substituted purine, 7-deaza-8-substituted purine, or an abasicnucleotide. For instance, a self-replicating RNA can include one or moremodified pyrimidine nucleobases, such as pseudouridine and/or5-methylcytosine residues. In some embodiments, however, the RNAincludes no modified nucleobases, and may include no modifiednucleotides i.e. all of the nucleotides in the RNA are standard A, C, Gand U ribonucleotides (except for any 5′ cap structure, which mayinclude a 7′-methylguanosine). In other embodiments, the RNA may includea 5′ cap comprising a 7′-methylguanosine, and the first 1, 2 or 3 5′ribonucleotides may be methylated at the 2′ position of the ribose.

A RNA used with the invention ideally includes only phosphodiesterlinkages between nucleosides, but in some embodiments it can containphosphoramidate, phosphorothioate, and/or methylphosphonate linkages.

Ideally, a liposome includes fewer than 10 different species of RNA e.g.5, 4, 3, or 2 different species; most preferably, a liposome includes asingle RNA species i.e. all RNA molecules in the liposome have the samesequence and same length.

The amount of RNA per liposome can vary. The number of individualself-replicating RNA molecules per liposome is typically ≤50 e.g. <20,<10, <5, or 1-4 per liposome.

The Immunogen

RNA molecules used with the invention encode a polypeptide immunogen.After administration of the liposomes the RNA is translated in vivo andthe immunogen can elicit an immune response in the recipient. Theimmunogen may elicit an immune response against a bacterium, a virus, afungus or a parasite (or, in some embodiments, against an allergen; andin other embodiments, against a tumor antigen). The immune response maycomprise an antibody response (usually including IgG) and/or acell-mediated immune response. The polypeptide immunogen will typicallyelicit an immune response which recognises the corresponding bacterial,viral, fungal or parasite (or allergen or tumour) polypeptide, but insome embodiments the polypeptide may act as a mimotope to elicit animmune response which recognises a bacterial, viral, fungal or parasitesaccharide. The immunogen will typically be a surface polypeptide e.g.an adhesin, a hemagglutinin, an envelope glycoprotein, a spikeglycoprotein, etc.

Self-replicating RNA molecules can encode a single polypeptide immunogenor multiple polypeptides. Multiple immunogens can be presented as asingle polypeptide immunogen (fusion polypeptide) or as separatepolypeptides. If immunogens are expressed as separate polypeptides thenone or more of these may be provided with an upstream IRES or anadditional viral promoter element. Alternatively, multiple immunogensmay be expressed from a polyprotein that encodes individual immunogensfused to a short autocatalytic protease (e.g. foot-and-mouth diseasevirus 2A protein), or as inteins.

Unlike references 1 and 14, the RNA encodes an immunogen. For theavoidance of doubt, the invention does not encompass RNA which encodes afirefly luciferase or which encodes a fusion protein of E. coliβ-galactosidase or which encodes a green fluorescent protein (GFP).Also, the RNA is not total mouse thymus RNA.

In some embodiments the immunogen elicits an immune response against oneof these bacteria:

-   -   Neisseria meningitidis: useful immunogens include, but are not        limited to, membrane proteins such as adhesins,        autotransporters, toxins, iron acquisition proteins, and factor        H binding protein. A combination of three useful polypeptides is        disclosed in reference 15.    -   Streptococcus pneumoniae: useful polypeptide immunogens are        disclosed in reference 16. These include, but are not limited        to, the RrgB pilus subunit, the beta-N-acetyl-hexosaminidase        precursor (spr0057), spr0096, General stress protein GSP-781        (spr2021, SP2216), serine/threonine kinase StkP (SP1732), and        pneumococcal surface adhesin PsaA.    -   Streptococcus pyogenes: useful immunogens include, but are not        limited to, the polypeptides disclosed in references 17 and 18.    -   Moraxella catarrhalis.    -   Bordetella pertussis: Useful pertussis immunogens include, but        are not limited to, pertussis toxin or toxoid (PT), filamentous        haemagglutinin (FHA), pertactin, and agglutinogens 2 and 3.    -   Staphylococcus aureus: Useful immunogens include, but are not        limited to, the polypeptides disclosed in reference 19, such as        a hemolysin, esxA, esxB, ferrichrome-binding protein (sta006)        and/or the sta011 lipoprotein.    -   Clostridium tetani: the typical immunogen is tetanus toxoid.    -   Cornynebacterium diphtheriae: the typical immunogen is        diphtheria toxoid.    -   Haemophilus influenzae: Useful immunogens include, but are not        limited to, the polypeptides disclosed in references 20 and 21.    -   Pseudomonas aeruginosa    -   Streptococcus agalactiae: useful immunogens include, but are not        limited to, the polypeptides disclosed in reference 17.    -   Chlamydia trachomatis: Useful immunogens include, but are not        limited to, PepA, LcrE, ArtJ, DnaK, CT398, OmpH-like, L7/L12,        OmcA, AtoS, CT547, Eno, HtrA and MurG (e.g. as disclosed in        reference 22. LcrE as disclosed in reference 23 and HtrA as        disclosed in reference 24 are two preferred immunogens.    -   Chlamydia pneumoniae: Useful immunogens include, but are not        limited to, the polypeptides disclosed in reference 25.    -   Helicobacter pylori: Useful immunogens include, but are not        limited to, CagA, VacA, NAP, and/or urease as disclosed in        reference 26.    -   Escherichia coli: Useful immunogens include, but are not limited        to, immunogens derived from enterotoxigenic E. coli (ETEC),        enteroaggregative E. coli (EAggEC), diffusely adhering E. coli        (DAEC), enteropathogenic E. coli (EPEC), extraintestinal        pathogenic E. coli (ExPEC) and/or enterohemorrhagic E. coli        (EHEC). ExPEC strains include uropathogenic E. coli (UPEC) and        meningitis/sepsis-associated E. coli (MNEC). Useful UPEC        polypeptide immunogens are disclosed in references 27 and 28.        Useful MNEC immunogens are disclosed in reference 29. A useful        immunogen for several E. coli types is AcfD as disclosed in        reference 30.    -   Bacillus anthracis    -   Yersinia pestis: Useful immunogens include, but are not limited        to, those disclosed in references 31 and 32.    -   Staphylococcus epidermis    -   Clostridium perfringens or Clostridium botulinums    -   Legionella pneumophila    -   Coxiella burnetii    -   Brucella, such as B.abortus, B.canis, B. melitensis, B.neotomae,        B.ovis, B.suis, B.pinmpediae.    -   Francisella, such as F.novicida, Fphilomiragia, F. tularensis.    -   Neisseria gonorrhoeae    -   Treponema pallidum    -   Haemophilus ducreyi    -   Enterococcus faecalis or Enterococcus faecium    -   Staphylococcus saprophyticus    -   Yersinia enterocolitica    -   Mycobacterium tuberculosis    -   Rickettsia    -   Listeria monocytogenes    -   Vibrio cholerae    -   Salmonella typhi    -   Borrelia burgdorferi    -   Porphyromonas gingivalis    -   Klebsiella

In some embodiments the immunogen elicits an immune response against oneof these viruses:

-   -   Orthomyxovirus: Useful immunogens can be from an influenza A, B        or C virus, such as the hemagglutinin, neuraminidase or matrix        M2 proteins. Where the immunogen is an influenza A virus        hemagglutinin it may be from any subtype e.g. H1, H2, H3, H4,        H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 or H16.    -   Paramyxoviridae viruses: Viral immunogens include, but are not        limited to, those derived from Pneumoviruses (e.g. respiratory        syncytial virus, RSV), Rubulaviruses (e.g. mumps virus),        Paramyxoviruses (e.g. parainfluenza virus), Metapneumoviruses        and Morbilliviruses (e.g. measles).    -   Poxviridae: Viral immunogens include, but are not limited to,        those derived from Orthopoxvirus such as Variola vera, including        but not limited to, Variola major and Variola minor.    -   Picornavirus: Viral immunogens include, but are not limited to,        those derived from Picornaviruses, such as Enteroviruses,        Rhinoviruses, Heparnavirus, Cardioviruses and Aphthoviruses. In        one embodiment, the enterovirus is a poliovirus e.g. a type 1,        type 2 and/or type 3 poliovirus. In another embodiment, the        enterovirus is an EV71 enterovirus. In another embodiment, the        enterovirus is a coxsackie A or B virus.    -   Bunyavirus: Viral immunogens include, but are not limited to,        those derived from an Orthobunyavirus, such as California        encephalitis virus, a Phlebovirus, such as Rift Valley Fever        virus, or a Nairovirus, such as Crimean-Congo hemorrhagic fever        virus.    -   Heparnavirus: Viral immunogens include, but are not limited to,        those derived from a Heparnavirus, such as hepatitis A virus        (HAV).    -   Filovirus: Viral immunogens include, but are not limited to,        those derived from a Filovirus, such as an Ebola virus        (including a Zaire, Ivory Coast, Reston or Sudan ebolavirus) or        a Marburg virus.    -   Togavirus: Viral immunogens include, but are not limited to,        those derived from a Togavirus, such as a Rubivirus, an        Alphavirus, or an Arterivirus. This includes rubella virus.    -   Flavivirus: Viral immunogens include, but are not limited to,        those derived from a Flavivirus, such as Tick-borne encephalitis        (TBE) virus, Dengue (types 1, 2, 3 or 4) virus, Yellow Fever        virus, Japanese encephalitis virus, Kyasanur Forest Virus, West        Nile encephalitis virus, St. Louis encephalitis virus, Russian        spring-summer encephalitis virus, Powassan encephalitis virus.    -   Pestivirus: Viral immunogens include, but are not limited to,        those derived from a Pestivirus, such as Bovine viral diarrhea        (BVDV), Classical swine fever (CSFV) or Border disease (BDV).    -   Hepadnavirus: Viral immunogens include, but are not limited to,        those derived from a Hepadnavirus, such as Hepatitis B virus. A        composition can include hepatitis B virus surface antigen        (HBsAg).    -   Other hepatitis viruses: A composition can include an immunogen        from a hepatitis C virus, delta hepatitis virus, hepatitis E        virus, or hepatitis G virus.    -   Rhabdovirus: Viral immunogens include, but are not limited to,        those derived from a Rhabdovirus, such as a Lyssavirus (e.g. a        Rabies virus) and Vesiculovirus (VSV).    -   Caliciviridae: Viral immunogens include, but are not limited to,        those derived from Calciviridae, such as Norwalk virus        (Norovirus), and Norwalk-like Viruses, such as Hawaii Virus and        Snow Mountain Virus.    -   Coronavirus: Viral immunogens include, but are not limited to,        those derived from a SARS coronavirus, avian infectious        bronchitis (IBV), Mouse hepatitis virus (MHV), and Porcine        transmissible gastroenteritis virus (TGEV). The coronavirus        immunogen may be a spike polypeptide.    -   Retrovirus: Viral immunogens include, but are not limited to,        those derived from an Oncovirus, a Lentivirus (e.g. HIV-1 or        HIV-2) or a Spumavirus.    -   Reovirus: Viral immunogens include, but are not limited to,        those derived from an Orthoreovirus, a Rotavirus, an Orbivirus,        or a Coltivirus.    -   Parvovirus: Viral immunogens include, but are not limited to,        those derived from Parvovirus B19.    -   Herpesvirus: Viral immunogens include, but are not limited to,        those derived from a human herpesvirus, such as, by way of        example only, Herpes Simplex Viruses (HSV) (e.g. HSV types 1 and        2), Varicella-zoster virus (VZV), Epstein-Barr virus (EBV),        Cytomegalovirus (CMV), Human Herpesvirus 6 (HHV6), Human        Herpesvirus 7 (HHV7), and Human Herpesvirus 8 (HHV8).    -   Papovaviruses: Viral immunogens include, but are not limited to,        those derived from Papillomaviruses and Polyomaviruses. The        (human) papillomavirus may be of serotype 1, 2, 4, 5, 6, 8, 11,        13, 16, 18, 31, 33, 35, 39, 41, 42, 47, 51, 57, 58, 63 or 65        e.g. from one or more of serotypes 6, 11, 16 and/or 18.    -   Adenovirus: Viral immunogens include those derived from        adenovirus serotype 36 (Ad-36).

In some embodiments, the immunogen elicits an immune response against avirus which infects fish, such as: infectious salmon anemia virus(ISAV), salmon pancreatic disease virus (SPDV), infectious pancreaticnecrosis virus (IPNV), channel catfish virus (CCV), fish lymphocystisdisease virus (FLDV), infectious hematopoietic necrosis virus (IHNV),koi herpesvirus, salmon picorna-like virus (also known as picorna-likevirus of atlantic salmon), landlocked salmon virus (LSV), atlanticsalmon rotavirus (ASR), trout strawberry disease virus (TSD), cohosalmon tumor o virus (CSTV), or viral hemorrhagic septicemia virus(VHSV).

Fungal immunogens may be derived from Dermatophytres, including:Epidermophyton floccusum, Microsporum audouini, Microsporum canis,Microsporum distortum, Microsporum equinum, Microsporum gypsum,Microsporum nanum, Trichophyton concentricum, Trichophyton equinum,Trichophyton gallinae, Trichophyton gypseum, Trichophyton megnini,Trichophyton mentagrophytes, Trichophyton quinckeanum, Trichophytonrubrum, Trichophyton schoenleini, Trichophyton tonsurans, Trichophytonverrucosum, T verrucosum var. album, var. discoides, var. ochraceum,Trichophyton violaceum, and/or Trichophyton faviforme; or fromAspergillus fumigatus, Aspergillus flavus, Aspergillus niger,Aspergillus nidulans, Aspergillus terreus, Aspergillus sydowi,Aspergillus flavatus, Aspergillus glaucus, Blastoschizomyces capitatus,Candida albicans, Candida enolase, Candida tropicalis, Candida glabrata,Candida krusei, Candida parapsilosis, Candida stellatoidea, Candidakusei, Candida parakwsei, Candida lusitaniae, Candida pseudotropicalis,Candida guilliermondi, Cladosporium carrionii, Coccidioides immitis,Blastomyces dermatidis, Cryptococcus neoformans, Geotrichum clavatum,Histoplasma capsulatum, Klebsiella pneumoniae, Microsporidia,Encephalitozoon spp., Septata intestinalis and Enterocytozoon bieneusi;the less common are Brachiola spp, Microsporidium spp., Nosema spp.,Pleistophora spp., Trachipleistophora spp., Vittaforma sppParacoccidioides brasiliensis, Pneumocystis carinii, Pythiumninsidiosum, Pityrosporum ovate, Sacharomyces cerevisae, Saccharomycesboulardii, Saccharomyces pombe, Scedosporium apiosperum, Sporothrixschenckii, Trichosporon beigelii, Toxoplasma gondii, Penicilliummarneffei, Malassezia spp., Fonsecaea spp., Wangiella spp., Sporothrixspp., Basidiobolus spp., Conidiobolus spp., Rhizopus spp, Mucor spp,Absidia spp, Mortierella spp, Cunninghamella spp, Saksenaea spp.,Alternaria spp, Curvularia spp, Helminthosporium spp, Fusarium spp,Aspergillus spp, Penicillium spp, Monolinia spp, Rhizoctonia spp,Paecilomyces spp, Pithomyces spp, and Cladosporium spp.

In some embodiments the immunogen elicits an immune response against aparasite from the Plasmodium genus, such as P.falciparum, P.vivax,P.malariae or P.ovale. Thus the invention may be used for immunisingagainst malaria. In some embodiments the immunogen elicits an immuneresponse against a parasite from the Caligidae family, particularlythose from the Lepeophtheirus and Caligus genera e.g. sea lice such asLepeophtheirus salmonis or Caligus rogercresseyi.

In some embodiments the immunogen elicits an immune response against:pollen allergens (tree-, herb, weed-, and grass pollen allergens);insect or arachnid allergens (inhalant, saliva and venom allergens, e.g.mite allergens, cockroach and midges allergens, hymenopthera venomallergens); animal hair and dandruff allergens (from e.g. dog, cat,horse, rat, mouse, etc.); and food allergens (e.g. a gliadin). Importantpollen allergens from trees, grasses and herbs are such originating fromthe taxonomic orders of Fagales, Oleales, Pinales and platanaceaeincluding, but not limited to, birch (Betula), alder (Alnus), hazel(Corylus), hornbeam (Carpinus) and olive (Olea), cedar (Cryptomeria andJuniperus), plane tree (Platanus), the order of Poales including grassesof the genera Lolium, Phleum, Poa, Cynodon, Dactylis, Holcus, Phalaris,Secale, and Sorghum, the orders of Asterales and Urticales includingherbs of the genera Ambrosia, Artemisia, and Parietaria. Other importantinhalation allergens are those from house dust mites of the genusDermatophagoides and Euroglyphus, storage mite e.g. Lepidoglyphys,Glycyphagus and Tyrophagus, those from cockroaches, midges and flease.g. Blatella, Periplaneta, Chironomus and Ctenocepphalides, and thosefrom mammals such as cat, dog and horse, venom allergens including suchoriginating from stinging or biting insects such as those from thetaxonomic order of Hymenoptera including bees (Apidae), wasps(Vespidea), and ants (Formicoidae).

In some embodiments the immunogen is a tumor antigen selected from: (a)cancer-testis antigens such as NY-ESO-1, SSX2, SCP1 as well as RAGE,BAGE, GAGE and MAGE family polypeptides, for example, GAGE-1, GAGE-2,MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6, and MAGE-12 (which canbe used, for example, to address melanoma, lung, head and neck, NSCLC,breast, gastrointestinal, and bladder tumors; (b) mutated antigens, forexample, p53 (associated with various solid tumors, e.g., colorectal,lung, head and neck cancer), p21/Ras (associated with, e.g., melanoma,pancreatic cancer and colorectal cancer), CDK4 (associated with, e.g.,melanoma), MUM1 (associated with, e.g., melanoma), caspase-8 (associatedwith, e.g., head and neck cancer), CIA 0205 (associated with, e.g.,bladder cancer), HLA-A2-R1701, beta catenin (associated with, e.g.,melanoma), TCR (associated with, e.g., T-cell non-Hodgkins lymphoma),BCR-abl (associated with, e.g., chronic myelogenous leukemia),triosephosphate isomerase, MA 0205, CDC-27, and LDLR-FUT; (c)over-expressed antigens, for example, Galectin 4 (associated with, e.g.,colorectal cancer), Galectin 9 (associated with, e.g., Hodgkin'sdisease), proteinase 3 (associated with, e.g., chronic myelogenousleukemia), WT 1 (associated with, e.g., various leukemias), carbonicanhydrase (associated with, e.g., renal cancer), aldolase A (associatedwith, e.g., lung cancer), PRAME (associated with, e.g., melanoma),HER-2/neu (associated with, e.g., breast, colon, lung and ovariancancer), mammaglobin, alpha-fetoprotein (associated with, e.g.,hepatoma), KSA (associated with, e.g., colorectal cancer), gastrin(associated with, e.g., pancreatic and gastric cancer), telomerasecatalytic protein, MUC-1 (associated with, e.g., breast and ovariancancer), G-250 (associated with, e.g., renal cell carcinoma), p53(associated with, e.g., breast, colon cancer), and carcinoembryonicantigen (associated with, e.g., breast cancer, lung cancer, and cancersof the gastrointestinal tract such as colorectal cancer); (d) sharedantigens, for example, melanoma-melanocyte differentiation antigens suchas MART-1/Melan A, gp100, MC1R, melanocyte-stimulating hormone receptor,tyrosinase, tyrosinase related protein-1/TRP1 and tyrosinase relatedprotein-2/TRP2 (associated with, e.g., melanoma); (e) prostateassociated antigens such as PAP, PSA, PSMA, PSH-P1, PSM-P1, PSM-P2,associated with e.g., prostate cancer; (f) immunoglobulin idiotypes(associated with myeloma and B cell lymphomas, for example). In certainembodiments, tumor immunogens include, but are not limited to, p15,Hom/Mel-40, H-Ras, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virusantigens, EBNA, human papillomavirus (HPV) antigens, including E6 andE7, hepatitis B and C virus antigens, human T-cell lymphotropic virusantigens, TSP-180, p185erbB2, p180erbB-3, c-met, mn-23H1, TAG-72-4, CA19-9, CA 72-4, CAM 17.1, NuMa, K-ras, p16, TAGE, PSCA, CT7, 43-9F, 5T4,791 Tgp72, beta-HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29\BCAA), CA195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, Ga733 (EpCAM),HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS1, SDCCAG16,TA-90 (Mac-2 binding protein/cyclophilin C-associated protein), TAAL6,TAG72, TLP, TPS, and the like.

Pharmaceutical Compositions

Liposomes of the invention are useful as components in pharmaceuticalcompositions for immunising subjects against various diseases. Thesecompositions will typically include a pharmaceutically acceptablecarrier in addition to the liposomes. A thorough discussion ofpharmaceutically acceptable carriers is available in reference 33.

A pharmaceutical composition of the invention may include one or moresmall molecule immunopotentiators. For example, the composition mayinclude a TLR2 agonist (e.g. Pam3CSK4), a TLR4 agonist (e.g. anaminoalkyl glucosaminide phosphate, such as E6020), a TLR7 agonist (e.g.imiquimod), a TLR8 agonist (e.g. resiquimod) and/or a TLR9 agonist (e.g.IC31). Any such agonist ideally has a molecular weight of <2000Da. Wherea RNA is encapsulated, in some embodiments such agonist(s) are alsoencapsulated with the RNA, but in other embodiments they areunencapsulated. Where a RNA is adsorbed to a particle, in someembodiments such agonist(s) are also adsorbed with the RNA, but in otherembodiments they are unadsorbed.

Pharmaceutical compositions of the invention may include the liposomesin plain water (e.g. w.f.i.) or in a buffer e.g. a phosphate buffer, aTris buffer, a borate buffer, a succinate buffer, a histidine buffer, ora citrate buffer. Buffer salts will typically be included in the 5-20 mMrange.

Pharmaceutical compositions of the invention may have a pH between 5.0and 9.5 e.g. between 6.0 and 8.0.

Compositions of the invention may include sodium salts (e.g. sodiumchloride) to give tonicity. A concentration of 10±2 mg/ml NaCl istypical e.g. about 9 mg/ml.

Compositions of the invention may include metal ion chelators. These canprolong RNA stability by removing ions which can acceleratephosphodiester hydrolysis. Thus a composition may include one or more ofEDTA, EGTA, BAPTA, pentetic acid, etc.. Such chelators are typicallypresent at between 10-500 μM e.g. 0.1 mM. A citrate salt, such as sodiumcitrate, can also act as a chelator, while advantageously also providingbuffering activity.

Pharmaceutical compositions of the invention may have an osmolality ofbetween 200 mOsm/kg and 400 mOsm/kg, e.g. between 240-360 mOsm/kg, orbetween 290-310 mOsm/kg.

Pharmaceutical compositions of the invention may include one or morepreservatives, such as thiomersal or 2-phenoxyethanol. Mercury-freecompositions are preferred, and preservative-free vaccines can beprepared.

Pharmaceutical compositions of the invention are preferably sterile.

Pharmaceutical compositions of the invention are preferablynon-pyrogenic e.g. containing <1 EU (endotoxin unit, a standard measure)per dose, and preferably <0.1 EU per dose.

Pharmaceutical compositions of the invention are preferably gluten free.

Pharmaceutical compositions of the invention may be prepared in unitdose form. In some embodiments a unit dose may have a volume of between0.1-1.0 ml e.g. about 0.5 ml.

The compositions may be prepared as injectables, either as solutions orsuspensions. The composition may be prepared for pulmonaryadministration e.g. by an inhaler, using a fine spray. The compositionmay be prepared for nasal, aural or ocular administration e.g. as sprayor drops. Injectables for intramuscular administration are typical.

Compositions comprise an immunologically effective amount of liposomes,as well as any other components, as needed. By ‘immunologicallyeffective amount’, it is meant that the administration of that amount toan individual, either in a single dose or as part of a series, iseffective for treatment or prevention. This amount varies depending uponthe health and physical condition of the individual to be treated, age,the taxonomic group of individual to be treated (e.g. non-human primate,primate, etc.), the capacity of the individual's immune system tosynthesise antibodies, the degree of protection desired, the formulationof the vaccine, the treating doctor's assessment of the medicalsituation, and other relevant factors. It is expected that the amountwill fall in a relatively broad range that can be determined throughroutine trials. The liposome and RNA content of compositions of theinvention will generally be expressed in terms of the amount of RNA perdose. A preferred dose has ≤100 μg RNA (e.g. from 10-100 μg, such asabout 10 μg, 25 μg, 50 μg, 75 μg or 100 μg), but expression can be seenat much lower levels e.g. ≤1 μg/dose, ≤100 ng/dose, ≤10 ng/dose, ≤1ng/dose, etc

The invention also provides a delivery device (e.g. syringe, nebuliser,sprayer, inhaler, dermal patch, etc.) containing a pharmaceuticalcomposition of the invention. This device can be used to administer thecomposition to a vertebrate subject.

Liposomes of the invention do not include ribosomes.

Methods of Treatment and Medical Uses

In contrast to the particles disclosed in reference 14, liposomes andpharmaceutical compositions of the invention are for in vivo use foreliciting an immune response against an immunogen of interest.

The invention provides a method for raising an immune response in avertebrate comprising the step of administering an effective amount of aliposome or pharmaceutical composition of the invention. The immuneresponse is preferably protective and preferably involves antibodiesand/or cell-mediated immunity. The method may raise a booster response.

The invention also provides a liposome or pharmaceutical composition ofthe invention for use in a method for raising an immune response in avertebrate.

The invention also provides the use of a liposome of the invention inthe manufacture of a medicament for raising an immune response in avertebrate.

By raising an immune response in the vertebrate by these uses andmethods, the vertebrate can be protected against various diseases and/orinfections e.g. against bacterial and/or viral diseases as discussedabove. The liposomes and compositions are immunogenic, and are morepreferably vaccine compositions. Vaccines according to the invention mayeither be prophylactic (i.e. to prevent infection) or therapeutic (i.e.to treat infection), but will typically be prophylactic.

The vertebrate is preferably a mammal, such as a human or a largeveterinary mammal (e.g. horses, cattle, deer, goats, pigs). Where thevaccine is for prophylactic use, the human is preferably a child (e.g. atoddler or infant) or a teenager; where the vaccine is for therapeuticuse, the human is preferably a teenager or an adult. A vaccine intendedfor children may also be administered to adults e.g. to assess safety,dosage, immunogenicity, etc.

Vaccines prepared according to the invention may be used to treat bothchildren and adults. Thus a human patient may be less than 1 year old,less than 5 years old, 1-5 years old, 5-15 years old, 15-55 years old,or at least 55 years old. Preferred patients for receiving the vaccinesare the elderly (e.g. ≥50 years old, ≥60 years old, and preferably ≥65years), the young (e.g. ≤5 years old), hospitalised patients, healthcareworkers, armed service and military personnel, pregnant women, thechronically ill, or immunodeficient patients. The vaccines are notsuitable solely for these groups, however, and may be used moregenerally in a population.

Compositions of the invention will generally be administered directly toa patient. Direct delivery may be accomplished by parenteral injection(e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly,intradermally, or to the interstitial space of a tissue; unlikereference 1, intraglossal injection is not typically used with thepresent invention). Alternative delivery routes include rectal, oral(e.g. tablet, spray), buccal, sublingual, vaginal, topical, transdermalor transcutaneous, intranasal, ocular, aural, pulmonary or other mucosaladministration. Intradermal and intramuscular administration are twopreferred routes. Injection may be via a needle (e.g. a hypodermicneedle), but needle-free injection may alternatively be used. A typicalintramuscular dose is 0.5 ml.

The invention may be used to elicit systemic and/or mucosal immunity,preferably to elicit an enhanced systemic and/or mucosal immunity.

Dosage can be by a single dose schedule or a multiple dose schedule.Multiple doses may be used in a primary immunisation schedule and/or ina booster immunisation schedule. In a multiple dose schedule the variousdoses may be given by the same or different routes e.g. a parenteralprime and mucosal boost, a mucosal prime and parenteral boost, etc.Multiple doses will typically be administered at least 1 week apart(e.g. about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, etc.). In oneembodiment, multiple doses may be administered approximately 6 weeks, 10weeks and 14 weeks after birth, e.g. at an age of 6 weeks, 10 weeks and14 weeks, as often used in the World Health Organisation's ExpandedProgram on Immunisation (“EPI”). In an alternative embodiment, twoprimary doses are administered about two months apart, e.g. about 7, 8or 9 weeks apart, followed by one or more booster doses about 6 monthsto 1 year after the second primary dose, e.g. about 6, 8, 10 or 12months after the second primary dose. In a further embodiment, threeprimary doses are administered about two months apart, e.g. about 7, 8or 9 weeks apart, followed by one or more booster doses about 6 monthsto 1 year after the third primary dose, e.g. about 6, 8, 10, or 12months after the third primary dose.

General

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, molecularbiology, immunology and pharmacology, within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., references34-40, etc.

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The term “about” in relation to a numerical value x is optional andmeans, for example, x±10%.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

References to charge, to cations, to anions, to zwitterions, etc., aretaken at pH 7.

TLR3 is the Toll-like receptor 3. It is a single membrane-spanningreceptor which plays a key role in the innate immune system. Known TLR3agonists include poly(I:C). “TLR3” is the approved HGNC name for thegene encoding this receptor, and its unique HGNC ID is HGNC:11849. TheRefSeq sequence for the human TLR3 gene is GI:2459625.

TLR7 is the Toll-like receptor 7. It is a single membrane-spanningreceptor which plays a key role in the innate immune system. Known TLR7agonists include e.g. imiquimod. “TLR7” is the approved HGNC name forthe gene encoding this receptor, and its unique HGNC ID is HGNC:15631.The RefSeq sequence for the human TLR7 gene is GI:67944638.

TLR8 is the Toll-like receptor 8. It is a single membrane-spanningreceptor which plays a key role in the innate immune system. Known TLR8agonists include e.g. resiquimod. “TLR8” is the approved HGNC name forthe gene encoding this receptor, and its unique HGNC ID is HGNC:15632.The RefSeq sequence for the human TLR8 gene is GI:20302165.

The RIG-I-like receptor (“RLR”) family includes various RNA helicaseswhich play key roles in the innate immune system as disclosed inreference 41. RLR-1 (also known as RIG-I or retinoic acid inducible geneI) has two caspase recruitment domains near its N-terminus. The approvedHGNC name for the gene encoding the RLR-1 helicase is “DDX58” (for DEAD(Asp-Glu-Ala-Asp) box polypeptide 58) and the unique HGNC ID isHGNC:19102. The RefSeq sequence for the human RLR-1 gene is GI:77732514.RLR-2 (also known as MDAS or melanoma differentiation-associated gene 5)also has two caspase recruitment domains near its N-terminus. Theapproved HGNC name for the gene encoding the RLR-2 helicase is “IFIH1”(for interferon induced with helicase C domain 1) and the unique HGNC IDis HGNC:18873. The RefSeq o sequence for the human RLR-2 gene is GI:27886567. RLR-3 (also known as LGP2 or laboratory of genetics andphysiology 2) has no caspase recruitment domains. The approved HGNC namefor the gene encoding the RLR-3 helicase is “DHX58” (for DEXH(Asp-Glu-X-His) box polypeptide 58) and the unique HGNC ID isHGNC:29517. The RefSeq sequence for the human RLR-3 gene isGI:149408121.

PKR is a double-stranded RNA-dependent protein kinase. It plays a keyrole in the innate immune system. “EIF2AK2” (for eukaryotic translationinitiation factor 2-alpha kinase 2) is the approved HGNC name for thegene encoding this enzyme, and its unique HGNC ID is HGNC:9437. TheRefSeq sequence for the human PKR gene is GI:208431825.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a gel with stained RNA. Lanes show (1) markers (2) nakedreplicon (3) replicon after RNase treatment (4) replicon encapsulated inliposome (5) liposome after RNase treatment (6) liposome treated withRNase then subjected to phenol/chloroform extraction.

FIG. 2 is an electron micrograph of liposomes.

FIG. 3 shows the structures of DLinDMA, DLenDMA and DODMA.

FIG. 4 shows a gel with stained RNA. Lanes show (1) markers (2) nakedreplicon (3) replicon encapsulated in liposome (4) liposome treated withRNase then subjected to phenol/chloroform extraction.

FIG. 5 shows protein expression at days 1, 3 and 6 after delivery of RNAas a virion-packaged replicon (squares), as naked RNA (diamonds), or inliposomes (+=0.1 μg, x=1 μg).

FIG. 6 shows protein expression at days 1, 3 and 6 after delivery offour different doses of liposome-encapsulated RNA.

FIG. 7 shows anti-F IgG titers in animals receiving virion-packagedreplicon (VRP or VSRP), 1 μg naked RNA, and 1 μg liposome-encapsulatedRNA.

FIG. 8 shows anti-F IgG titers in animals receiving VRP, 1 μg naked RNA,and 0.1 g or 1 μg liposome-encapsulated RNA.

FIG. 9 shows neutralising antibody titers in animals receiving VRP oreither 0.1 g or 1 μg liposome-encapsulated RNA.

FIG. 10 shows expression levels after delivery of a replicon as nakedRNA (circles), liposome-encapsulated RNA (triangle & square), or as alipoplex (inverted triangle).

FIG. 11 shows F-specific IgG titers (2 weeks after second dose) afterdelivery of a replicon as naked RNA (0.01-1 μg), liposome-encapsulatedRNA (0.01-10 μg), or packaged as a virion (VRP, 10⁶ infectious units orIU).

FIG. 12 shows F-specific IgG titers (circles) and PRNT titers (squares)after delivery of a replicon as naked RNA (1 μg), liposome-encapsulatedRNA (0.1 or 1 μg), or packaged as a virion (VRP, 10⁶ IU). Titers innaïve mice are also shown. Solid lines show geometric means.

FIG. 13 shows intracellular cytokine production after restimulation withsynthetic peptides representing the major epitopes in the F protein, 4weeks after a second dose. The y-axis shows the % cytokine+of CD8+CD4−.

FIGS. 14A and 14B show F-specific IgG titers (mean logio titers±std dev)over 63 days (FIG. 14A) and 210 days (FIG. 14B) after immunisation ofcalves. The three lines are easily distinguished at day 63 and are, frombottom to top: PBS negative control; liposome-delivered RNA; and the“Triangle 4” product.

FIG. 15 shows SEAP expression (relative intensity) at day 6 against pKaof lipids used in the liposomes. Circles show levels for liposomes withDSPC, and squares for liposomes without DSPC; sometimes a square andcircle overlap, leaving only the square visible for a given pKa.

FIG. 16 shows anti-F titers expression (relative to RV01, 100%) twoweeks after a first dose of replicon encoding F protein. The titers areplotted against pKa in the same way as in FIG. 15. The star shows RV02,which used a cationic lipid having a higher pKa than the other lipids.Triangles show data for liposomes lacking DSPC; circles are forliposomes which included DSPC.

FIG. 17 shows total IgG titers after replicon delivery in liposomesusing, from left to right, RV01, RV16, RV17, RV18 or RV19. Bars showmeans. The upper bar in each case is 2wp2 (i.e. 2 weeks after seconddose), whereas the lower bar is 2wpl.

FIG. 18 shows IgG titers in 13 groups of mice. Each circle is anindividual mouse, and solid lines show geometric means. The dottedhorizontal line is the assay's detection limit. The 13 groups are, fromleft to right, A to M as described below.

FIGS. 19A and 19B show IL-6 (FIG. 19A) and IFNa (FIG. 19B) (pg/ml)released by pDC. There are 4 pairs of bars, from left to right: control;immunised with RNA+DOTAP; immunised with RNA+lipofectamine; andimmunised with RNA in liposomes. In each pair the black bar is wild-typemice, grey is rsql mutant.

MODES FOR CARRYING OUT THE INVENTION RNA Replicons

Various replicons are used below. In general these are based on a hybridalphavirus genome with non-structural proteins from venezuelan equineencephalitis virus (VEEV), a packaging signal from sindbis virus, and a3′ UTR from Sindbis virus or a VEEV mutant. The replicon is about 10kblong and has a poly-A tail.

Plasmid DNA encoding alphavirus replicons (named: pT7-mVEEV-FL.RSVF orA317; pT7-mVEEV-SEAP or A306; pSP6-VCR-GFP or A50) served as a templatefor synthesis of RNA in vitro. The replicons contain the alphavirusgenetic elements required for RNA replication but lack those encodinggene products necessary for particle assembly; the structural proteinsare instead replaced by a protein of interest (either a reporter, suchas SEAP or GFP, or an immunogen, such as full-length RSV F protein) andso the replicons are incapable of inducing the generation of infectiousparticles. A bacteriophage (T7 or SP6) promoter upstream of thealphavirus cDNA facilitates the synthesis of the replicon RNA in vitroand a hepatitis delta virus (HDV) ribozyme immediately downstream of thepoly(A)-tail generates the correct 3′-end through its self-cleavingactivity.

Following linearization of the plasmid DNA downstream of the HDVribozyme with a suitable restriction endonuclease, run-off transcriptswere synthesized in vitro using T7 or SP6 bacteriophage derivedDNA-dependent RNA polymerase. Transcriptions were performed for 2 hoursat 37° C. in the presence of 7.5 mM (T7 RNA polymerase) or 5 mM (SP6 RNApolymerase) of each of the nucleoside triphosphates (ATP, CTP, GTP andUTP) following the instructions provided by the manufacturer (Ambion).Following transcription the template DNA was digested with TURBO DNase(Ambion). The replicon RNA was precipitated with LiC1 and reconstitutedin nuclease-free water. Uncapped RNA was capped post-transcriptionallywith Vaccinia Capping Enzyme (VCE) using the ScriptCap m7G CappingSystem (Epicentre Biotechnologies) as outlined in the user manual;replicons capped in this way are given the “v” prefix e.g. vA317 is theA317 replicon capped by VCE. Post-transcriptionally capped RNA wasprecipitated with LiCl and reconstituted in nuclease-free water. Theconcentration of the RNA samples was determined by measuring OD_(260nm).Integrity of the in vitro transcripts was confirmed by denaturingagarose gel electrophoresis.

Liposomal Encapsulation

RNA was encapsulated in liposomes made by the method of references 11and 42. The liposomes were made of 10% DSPC (zwitterionic), 40% DLinDMA(cationic), 48% cholesterol and 2% PEG-conjugated DMG (2kDa PEG). Theseproportions refer to the % moles in the total liposome.

DLinDMA (1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane) was synthesizedusing the procedure of reference 6. DSPC(1,2-Diastearoyl-sn-glycero-3-phosphocholine) was purchased fromGenzyme. Cholesterol was obtained from Sigma-Aldrich. PEG-conjugated DMG(1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol), ammonium salt), DOTAP(1,2-dioleoyl-3-trimethylammonium-propane, chloride salt) and DC-chol(3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride)were from Avanti Polar Lipids.1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol was obtainedfrom NOF Corporation (catalog # GM-020).

Briefly, lipids were dissolved in ethanol (2 ml), a RNA replicon wasdissolved in buffer (2 ml, 100 mM sodium citrate, pH 6) and these weremixed with 2 ml of buffer followed by 1 hour of equilibration. Themixture was diluted with 6 ml buffer then filtered. The resultingproduct contained liposomes, with ˜95% encapsulation efficiency.

For example, in one particular method, fresh lipid stock solutions wereprepared in ethanol. 37 mg of DLinDMA, 11.8 mg of DSPC, 27.8 mg ofcholesterol and 8.07 mg of PEG-DMG were weighed and dissolved in 7.55 mLof ethanol. The freshly prepared lipid stock solution was gently rockedat 37° C. for about 15 min to form a homogenous mixture. Then, 755 μL ofthe stock was added to 1.245 mL ethanol to make a working lipid stocksolution of 2 mL. This amount of lipids was used to form liposomes with250 μg RNA. A 2 mL working solution of RNA was also prepared from astock solution of ˜1 μg/μL in 100 mM citrate buffer (pH 6). Three 20 mLglass vials (with stir bars) were rinsed with RNase Away solution(Molecular BioProducts) and washed with plenty of MilliQ water beforeuse to decontaminate the vials of RNases. One of the vials was used forthe RNA working solution and the others for collecting the lipid and RNAmixes (as described later). The working lipid and RNA solutions wereheated at 37° C. for 10 min before being loaded into 3 cc luer-loksyringes. 2 mL citrate buffer (pH 6) was loaded in another 3 cc syringe.Syringes containing RNA and the lipids were connected to a T mixer(PEEK™ 500 μm ID junction, Idex Health Science) using FEP tubing(fluorinated ethylene-propylene; all FEP tubing used has a 2 mm internaldiameter and a 3 mm outer diameter). The outlet from the T mixer wasalso FEP tubing. The third syringe containing the citrate buffer wasconnected to a separate piece of FEP tubing. All syringes were thendriven at a flow rate of 7 mL/min using a syringe pump. The tube outletswere positioned to collect the mixtures in a 20 mL glass vial (whilestirring). The stir bar was taken out and the ethanol/aqueous solutionwas allowed to equilibrate to room temperature for 1 h. 4 ml of themixture was loaded into a 5 cc syringe, which was connected to a pieceof FEP tubing and in another 5 cc syringe connected to an equal lengthof FEP tubing, an equal amount of 100 mM citrate buffer (pH 6) wasloaded. The two syringes were driven at 7 mL/min flow rate using thesyringe pump and the final mixture collected in a 20 mL glass vial(while stirring). Next, the mixture collected from the second mixingstep (liposomes) were passed through a Mustang Q membrane (ananion-exchange support that binds and removes anionic molecules,obtained from Pall Corporation). Before using this membrane for theliposomes, 4 mL of 1 M NaOH, 4 mL of 1 M NaCl and 10 mL of 100 mMcitrate buffer (pH 6) were successively passed through it. Liposomeswere warmed for 10 min at 37° C. before passing through the membrane.Next, liposomes were concentrated to 2 mL and dialyzed against 10-15volumes of 1×PBS using by tangential flow filtration before recoveringthe final product. The TFF system and hollow fiber filtration membraneswere purchased from Spectrum Labs (Rancho Dominguez) and were usedaccording to the manufacturer's guidelines. Polysulfone hollow fiberfiltration membranes with a 100 kD pore size cutoff and 8 cm² surfacearea were used. For in vitro and in vivo experiments formulations werediluted to the required RNA concentration with 1×PBS.

FIG. 2 shows an example electron micrograph of liposomes prepared bythese methods. These liposomes contain encapsulated RNA encodingfull-length RSV F antigen. Dynamic light scattering of one batch showedan average diameter of 141nm (by intensity) or 78 nm (by number).

The percentage of encapsulated RNA and RNA concentration were determinedby Quant-iT RiboGreen RNA reagent kit (Invitrogen), followingmanufacturer's instructions. The ribosomal RNA standard provided in thekit was used to generate a standard curve. Liposomes were diluted 10× or100× in 1×TE buffer (from kit) before addition of the dye. Separately,liposomes were diluted 10× or 100× in 1×TE buffer containing 0.5% TritonX before addition of the dye (to disrupt the liposomes and thus to assaytotal RNA). Thereafter an equal amount of dye was added to each solutionand then ˜180 μL of each solution after dye addition was loaded induplicate into a 96 well tissue culture plate. The fluorescence (Ex 485nm, Em 528 nm) was read on a microplate reader. All liposomeformulations were dosed in vivo based on the encapsulated amount of RNA.

Encapsulation in liposomes was shown to protect RNA from RNasedigestion. Experiments used 3.8 mAU of RNase A per microgram of RNA,incubated for 30 minutes at room temperature. RNase was inactivated withProteinase K at 55° C. for 10 minutes. A 1:1 v/v mixture of sample to25:24:1 v/v/v, phenol:chloroform:isoamyl alcohol was then added toextract the RNA from the lipids into the aqueous phase. Samples weremixed by vortexing for a few seconds and then placed on a centrifuge for15 minutes at 12 k RPM. The aqueous phase (containing the RNA) wasremoved and used to analyze the RNA. Prior to loading (400 ng RNA perwell) all the samples were incubated with formaldehyde loading dye,denatured for 10 minutes at 65° C. and cooled to room temperature.Ambion Millennium markers were used to approximate the molecular weightof the RNA construct. The gel was run at 90 V. The gel was stained using0.1% SYBR gold according to the manufacturer's guidelines in water byrocking at room temperature for 1 hour. FIG. 1 shows that RNasecompletely digests RNA in the absence of encapsulation (lane 3). RNA isundetectable after encapsulation (lane 4), and no change is seen ifthese liposomes are treated with RNase (lane 4). After RNase-treatedliposomes are subjected to phenol extraction, undigested RNA is seen(lane 6). Even after 1 week at 4° C. the RNA could be seen without anyfragmentation (FIG. 4, arrow). Protein expression in vivo was unchangedafter 6 weeks at 4° C. and one freeze-thaw cycle. Thusliposome-encapsulated RNA is stable.

To assess in vivo expression of the RNA a reporter enzyme (SEAP;secreted alkaline phosphatase) was encoded in the replicon, rather thanan immunogen. Expression levels were measured in sera diluted 1:4 in 1×Phospha-Light dilution buffer using a chemiluminescent alkalinephosphate substrate. 8-10 week old BALB/c mice (5/group) were injectedintramuscularly on day 0, 50μ1 per leg with 0.1 μg or 1 μg RNA dose. Thesame vector was also administered without the liposomes (in RNase free1×PBS) at lug. Virion-packaged replicons were also tested.Virion-packaged replicons used herein (referred to as “VRPs”) wereobtained by the methods of reference 43, where the alphavirus repliconis derived from the mutant VEEV or a chimera derived from the genome ofVEEV engineered to contain the 3′ UTR of Sindbis virus and a Sindbisvirus packaging signal (PS), packaged by co-electroporating them intoBHK cells with defective helper RNAs encoding the Sindbis virus capsidand glycoprotein genes.

As shown in FIG. 5, encapsulation increased SEAP levels by about ½ logat the lug dose, and at day 6 expression from a 0.1 μg encapsulated dosematched levels seen with lug unencapsulated dose. By day 3 expressionlevels exceeded those achieved with VRPs (squares). Thus expressedincreased when the RNA was formulated in the liposomes relative to thenaked RNA control, even at a 10× lower dose. Expression was also higherrelative to the VRP control, but the kinetics of expression were verydifferent (see FIG. 5). Delivery of the RNA with electroporationresulted in increased expression relative to the naked RNA control, butthese levels were lower than with liposomes.

To assess whether the effect seen in the liposome groups was due merelyto the liposome components, or was linked to the encapsulation, thereplicon was administered in encapsulated form (with two differentpurification protocols, 0.1 μg RNA), or mixed with the liposomes aftertheir formation (a non-encapsulated “lipoplex”, 0.1 μg RNA), or as nakedRNA (1 μg). FIG. 10 shows that the lipoplex gave the lowest levels ofexpression, showing that shows encapsulation is essential for potentexpression.

In vivo studies using liposomal delivery confirmed these findings. Micereceived various combinations of (i) self-replicating RNA repliconencoding full-length RSV F protein (ii) self-replicating GFP-encodingRNA replicon (iii) GFP-encoding RNA replicon with a knockout in nsP4which eliminates self-replication (iv) full-length RSV F-protein. 13groups in total received:

A — — B 0.1 μg of (i), naked — C 0.1 μg of (i), encapsulated in liposome— D 0.1 μg of (i), with separate liposomes — E 0.1 μg of (i), naked 10μg of (ii), naked F 0.1 μg of (i), naked 10 μg of (iii), naked G 0.1 μgof (i), encapsulated in liposome 10 μg of (ii), naked H 0.1 μg of (i),encapsulated in liposome 10 μg of (iii), naked I 0.1 μg of (i),encapsulated in liposome 1 μg of (ii), encapsulated in liposome J 0.1 μgof (i), encapsulated in liposome 1 μg of (iii), encapsulated in liposomeK 5 μg F protein — L 5 μg F protein 1 μg of (ii), encapsulated inliposome M 5 μg F protein 1 μg of (iii), encapsulated in liposome

Results in FIG. 18 show that F-specific IgG responses requiredencapsulation in the liposome rather than mere co-delivery (comparegroups C & D). A comparison of groups K, L and M shows that the RNAprovided an adjuvant effect against co-delivered protein, and thiseffect was seen with both replicating and non-replicating RNA.

Further SEAP experiments showed a clear dose response in vivo, withexpression seen after delivery of as little as ing RNA (FIG. 6). Furtherexperiments comparing expression from encapsulated and naked repliconsindicated that 0.01 μg encapsulated RNA was equivalent to 1 μg of nakedRNA. At a 0.5 μg dose of RNA the encapsulated material gave a 12-foldhigher expression at day 6; at a 0.1 μg dose levels were 24-fold higherat day 6.

Rather than looking at average levels in the group, individual animalswere also studied. Whereas several animals were non-responders to nakedreplicons, encapsulation eliminated non-responders.

Further experiments replaced DLinDMA with DOTAP. Although the DOTAPliposomes gave better expression than naked replicon, they were inferiorto the DLinDMA liposomes (2- to 3-fold difference at day 1). WhereasDOTAP has a quaternary amine, and so have a positive charge at the pointof delivery, DLinDMA has a tertiary amine.

To assess in vivo immunogenicity a replicon was constructed to expressfull-length F protein from respiratory syncytial virus (RSV). This wasdelivered naked (1 μg), encapsulated in liposomes (0.1 or 1 μg), orpackaged in virions (10⁶ IU; “VRP”) at days 0 and 21. FIG. 7 showsanti-F IgG titers 2 weeks after the second dose, and the liposomesclearly enhance immunogenicity. FIG. 8 shows titers 2 weeks later, bywhich point there was no statistical difference between the encapsulatedRNA at 0.1 μg, the encapsulated RNA at 1 μg, or the VRP group.Neutralisation titers (measured as 60% plaque reduction, “PRNT60”) werenot significantly different in these three groups 2 weeks after thesecond dose (FIG. 9). FIG. 12 shows both IgG and PRNT titers 4 weeksafter the second dose.

FIG. 13 confirms that the RNA elicits a robust CD8 T cell response.

Further experiments compared F-specific IgG titers in mice receivingVRP, 0.1 μg liposome-encapsulated RNA, or 1 μg liposome-encapsulatedRNA. Titer ratios (VRP:liposome) at various times after the second dosewere as follows:

2 weeks 4 weeks 8 weeks 0.1 μg 2.9 1.0 1.1   1 μg 2.3 0.9 0.9

Thus the liposome-encapsulated RNA induces essentially the samemagnitude of immune response as seen with virion delivery.

Further experiments showed superior F-specific IgG responses with a 10μg dose, equivalent responses for 1 μg and 0.1 μg doses, and a lowerresponse with a 0.01 μg dose. FIG. 11 shows IgG titers in mice receivingthe replicon in naked form at 3 different doses, in liposomes at 4different doses, or as VRP (10⁶ IU). The response seen with 1 μgliposome-encapsulated RNA was statistically insignificant (ANOVA) whencompared to VRP, but the higher response seen with 10 μgliposome-encapsulated RNA was statistically significant (p<0.05) whencompared to both of these groups.

A further study confirmed that the 0.1 μg of liposome-encapsulated RNAgave much higher anti-F IgG responses (15 days post-second dose) than0.1 μg of delivered DNA, and even was more immunogenic than 20 μgplasmid DNA encoding the F antigen, delivered by electroporation (Elgen™DNA Delivery System, Inovio).

A further study was performed in cotton rats (Sigmodon hispidis) insteadof mice. At a 1 μg dose liposome encapsulation increased F-specific IgGtiters by 8.3-fold compared to naked RNA and increased PRNT titers by9.5-fold. The magnitude of the antibody response was equivalent to thatinduced by 5×10⁶ IU VRP. Both naked and liposome-encapsulated RNA wereable to protect the cotton rats from RSV challenge (1×10⁵ plaque formingunits), reducing lung viral load by at least 3.5 logs. Encapsulationincreased the reduction by about 2-fold.

A large-animal study was performed in cattle. Cows were immunised with66 μg of replicon encoding full-length RSV F protein at days 0 and 21,formulated inside liposomes. PBS alone was used as a negative control,and a licensed vaccine was used as a positive control (“Triangle 4” fromFort Dodge, containing killed virus). FIGS. 14 show F-specific IgGtiters over 63 day and 210 day periods, respectively, starting from thefirst immunisation. The RNA replicon was immunogenic in the cows,although it gave lower titers than the licensed vaccine. All vaccinatedcows showed F-specific antibodies after the second dose, and titers werevery stable from the period of 2 to 6 weeks after the second dose (andwere particularly stable for the RNA vaccine).

Mechanism of Action

Bone marrow derived dendritic cells (pDC) were obtained from wild-typemice or the “Resq” (rsql) mutant strain. The mutant strain has a pointmutation at the amino terminus of its TLR7 receptor which abolishes TLR7signalling without affecting ligand binding as disclosed in reference44. The cells were stimulated with replicon RNA formulated with DOTAP,lipofectamine 2000 or inside a liposome. As shown in FIGS. 19A and 19B,IL-6 and INFα, respectively, were induced in WT cells but this responsewas almost completely abrogated in mutant mice. These results shows thatTLR7 is required for RNA recognition in immune cells, and thatliposome-encapsulated replicons can cause immune cells to secrete highlevels of both interferons and pro-inflammatory cytokines.

pKa Measurement

The pKa of a lipid is measured in water at standard temperature andpressure using the following technique:

-   -   2 mM solution of lipid in ethanol is prepared by weighing the        lipid and dissolving in ethanol. 0.3 mM solution of fluorescent        probe 6-(p-Toluidino)-2-naphthalenesulfonic acid (TNS) in        ethanol:methanol 9:1 is prepared by first making 3 mM solution        of TNS in methanol and then diluting to 0.3 mM with ethanol.    -   An aqueous buffer containing sodium phosphate, sodium citrate        sodium acetate and sodium chloride, at the concentrations 20 mM,        25 mM, 20 mM and 150 mM, respectively, is prepared. The buffer        is split into eight parts and the pH adjusted either with 12N        HCl or 6N NaOH to 4.44-4.52, 5.27, 6.15-6.21, 6.57, 7.10-7.20,        7.72-7.80, 8.27-8.33 and 10.47-11.12. 400 μL of 2 mM lipid        solution and 800 μL of 0.3 mM TNS solution are mixed.    -   7.5 μL of probe/lipid mix are added to 242.5 μL of buffer in a 1        mL 96 well plate. This is done with all eight buffers. After        mixing, 100 μL of each probe/lipid/buffer mixture is transferred        to a 250 μL black with clear bottom 96 well plate (e.g. model        COSTAR 3904, Corning). A convenient way of performing this        mixing is to use the Tecan Genesis RSP150 high throughput liquid        handler and Gemini Software.    -   Fluorescence of each probe/lipid/buffer mixture is measured        (e.g. with a SpectraMax M5 spectrophotometer and SoftMax pro 5.2        software) with 322 nm excitation, 431 nm emission (auto cutoff        at 420 nm).    -   After the measurement, the background fluorescence value of an        empty well on the 96 well plate is subtracted from each        probe/lipid/buffer mixture. The fluorescence intensity values        are then normalized to the value at lowest pH. The normalized        fluorescence intensity is then plotted against pH and a line of        best fit is provided.    -   The point on the line of best fit at which the normalized        fluorescence intensity is equal to 0.5 is found. The pH        corresponding to normalized fluorescence intensity equal to 0.5        is found and is considered the pKa of the lipid.

This method gives a pKa of 5.8 for DLinDMA. The pKa values measured bythis method for cationic lipids of reference 5 are included below.

Encapsulation in Liposomes Using Alternative Cationic Lipids

As an alternative to using DlinDMA, the cationic lipids of reference 5are used. These lipids can be synthesised as disclosed in reference 5.

The liposomes formed above using DlinDMA are referred to hereafter asthe “RV01” series. The DlinDMA was replaced with various cationic lipidsin series “RV02” to “RV12” as described below. Two different types ofeach liposome were formed, using 2% PEG2000-DMG with either (01) 40% ofthe cationic lipid, 10% DSPC, and 48% cholesterol, or (02) 60% of thecationic lipid and 38% cholesterol. Thus a comparison of the (01) and(02) liposomes shows the effect of the neutral zwitterionic lipid.

RV02 liposomes were made using the following cationic lipid (pKa>9,without a tertiary amine):

RV03 liposomes were made using the following cationic lipid (pKa 6.4):

RV04 liposomes were made using the following cationic lipid (pKa 6.62):

RV05 liposomes were made using the following cationic lipid (pKa 5.85):

RV06 liposomes were made using the following cationic lipid (pKa 7.27):

RV07 liposomes were made using the following cationic lipid (pKa 6.8):

RV08 liposomes were made using the following cationic lipid (pKa 5.72):

RV09 liposomes were made using the following cationic lipid (pKa 6.07):

RV10 liposomes were made for comparison using the following cationiclipid (pKa 7.86):

RV11 liposomes were made using the following cationic lipid (pKa 6.41):

RV12 liposomes were made using the following cationic lipid (pKa 7):

RV16 liposomes were made using the following cationic lipid (pKa 6.1) asdisclosed in reference 45:

RV17 liposomes were made using the following cationic lipid (pKa 6.1) asdisclosed in reference 45:

RV18 liposomes were made using DODMA. RV19 liposomes were made usingDOTMA, and RV13 liposomes were made with DOTAP, both having a quaternaryamine headgroup.

These liposomes were characterised and were tested with the SEAPreporter described above. The following table shows the size of theliposomes (Z average and polydispersity index), the % of RNAencapsulation in each liposome, together with the SEAP activity detectedat days 1 and 6 after injection. SEAP activity is relative to “RV01(02)”liposomes made from DlinDMA, cholesterol and PEG-DMG:

Lipid % SEAP SEAP RV pKa Zav (pdI) encapsulation day 1 day 6 RV01 (01)5.8 154.6 (0.131) 95.5 80.9 71.1 RV01 (02) 5.8 162.0 (0.134) 85.3 100100 RV02 (01) >9 133.9 (0.185) 96.5 57 45.7 RV02 (02) >9 134.6 (0.082)97.6 54.2 4.3 RV03 (01) 6.4 158.3 (0.212) 62.0 65.7 44.9 RV03 (02) 6.4164.2 (0.145) 86 62.2 39.7 RV04 (01) 6.62 131.0 (0.145) 74.0 91 154.8RV04 (02) 6.62 134.6 (0.117) 81.5 90.4 142.6 RV05 (01) 5.85 164.0(0.162) 76.0 76.9 329.8 RV05 (02) 5.85 177.8 (0.117) 72.8 67.1 227.9RV06 (01) 7.27 116.0 (0.180) 79.8 25.5 12.4 RV06 (02) 7.27 136.3 (0.164)74.9 24.8 23.1 RV07 (01) 6.8 140.6 (0.184) 77 26.5 163.3 RV07 (02) 6.8138.6 (0.122) 87 29.7 74.8 RV08 (01) 5.72 176.7 (0.185) 50 76.5 187 RV08(02) 5.72 199.5 (0.191) 46.3 82.4 329.8 RV09 (01) 6.07 165.3 (0.169)72.2 65.1 453.9 RV09 (02) 6.07 179.5 (0.157) 65 68.5 658.2 RV10 (01)7.86 129.7 (0.184) 78.4 113.4 47.8 RV10 (02) 7.86 147.6 (0.131) 80.978.2 10.4 RV11 (01) 6.41 129.2 (0.186) 71 113.6 242.2 RV11 (02) 6.41 139 (0198) 75.2 71.8 187.2 RV12 (01) 7 135.7 (0.161) 78.8 65 10 RV12(02) 7 158.3 (0.287) 69.4 78.8 8.2

FIG. 15 plots the SEAP levels at day 6 against the pKa of the cationiclipids. The best results are seen where the lipid has a pKa between 5.6and 6.8, and ideally between 5.6 and 6.3.

These liposomes were also used to deliver a replicon encodingfull-length RSV F protein. Total IgG titers against F protein two weeksafter the first dose (2wp1) are plotted against pKa in FIG. 16. The bestresults are seen where the pKa is where the cationic lipid has a pKabetween 5.7-5.9, but pKa alone is not enough to guarantee a high titere.g. the lipid must still support liposome formation.

RSV Immunogenicity

Further work was carried out with a self-replicating replicon (vA317)encoding RSV F protein. BALB/c mice, 4 or 8 animals per group, weregiven bilateral intramuscular vaccinations (50 μL per leg) on days 0 and21 with the replicon (1 μg) alone or formulated as liposomes with theRV01 or RVO5 lipids (see above; pKa of 5.8 or 5.85) or with RV13. TheRV01 liposomes had 40% DlinDMA, 10% DSPC, 48% cholesterol and 2%PEG-DMG, but with differing amounts of RNA. The RV05(01) liposomes had40% cationic lipid, 48% cholesterol, 10% DSPC, and 2% PEG-DMG; theRV05(02) liposomes had 60% cationic lipid, 38% cholesterol, and 2%PEG-DMG. The RV13 liposomes had 40% DOTAP, 10% DPE, 48% cholesterol and2% PEG-DMG. For comparison, naked plasmid DNA (20 μg) expressing thesame RSV-F antigen was delivered either using electroporation or withRV01(10) liposomes (0.1μg DNA). Four mice were used as a naive controlgroup.

Liposomes were prepared by method (A) or method (B). In method (A) freshlipid stock solutions in ethanol were prepared. 37 mg of cationic lipid,11.8 mg of DSPC, 27.8 mg of cholesterol and 8.07 mg of PEG-DMG wereweighed and dissolved in 7.55 mL of ethanol. The freshly prepared lipidstock solution was gently rocked at 37° C. for about 15 min to form ahomogenous mixture. Then, 226.7 μL of the stock was added to 1.773 mLethanol to make a working lipid stock solution of 2 mL. This amount oflipids was used to form liposomes with 75 μg RNA to give an 8:1 nitrogento phosphate ratio (except that in RV01 (08) and RV01 (09) this ratiowas modified to 4:1 or 16:1). A 2 mL working solution of RNA (or, forRV01(10), DNA) was also prepared from a stock solution of ˜1 μg/μL in100 mM citrate buffer (pH 6). Three 20 mL glass vials (with stir bars)were rinsed with RNase Away solution (Molecular BioProducts) and washedwith plenty of MilliQ water before use to decontaminate the vials ofRNases. One of the vials was used for the RNA working solution and theothers for collecting the lipid and RNA mixes (as described later). Theworking lipid and RNA solutions were heated at 37° C. for 10 min beforebeing loaded into 3cc syringes. 2 mL of citrate buffer (pH 6) was loadedin another 3 cc syringe. Syringes containing RNA and the lipids wereconnected to a T mixer (PEEK™ 500 μm ID junction) using FEP tubing. Theoutlet from the T mixer was also FEP tubing. The third syringecontaining the citrate buffer was connected to a separate piece of FEPtubing. All syringes were then driven at a flow rate of 7 mL/min using asyringe pump. The tube outlets were positioned to collect the mixturesin a 20 mL glass vial (while stirring). The stir bar was taken out andthe ethanol/aqueous solution was allowed to equilibrate to roomtemperature for 1 hour. Then the mixture was loaded in a 5 cc syringe,which was fitted to a piece of FEP tubing and in another 5 cc syringewith equal length of FEP tubing, an equal volume of 100 mM citratebuffer (pH 6) was loaded. The two syringes were driven at 7 mL/min flowrate using a syringe pump and the final mixture collected in a 20 mLglass vial (while stirring). Next, liposomes were concentrated to 2 mLand dialyzed against 10-15 volumes of 1×PBS using TFF before recoveringthe final product. The TFF system and hollow fiber filtration membraneswere purchased from Spectrum Labs and were used according to themanufacturer's guidelines. Polyethersulfone (PES) hollow fiberfiltration membranes (part number P-C1-100E-100-01N) with a 100 kD poresize cutoff and 20 cm² surface area were used. For in vitro and in vivoexperiments, formulations were diluted to the required RNA concentrationwith 1X×PBS.

Preparation method (B) differed in two ways from method (A). Firstly,after collection in the 20 mL glass vial but before TFF concentration,the mixture was passed through a Mustang Q membrane (an anion-exchangesupport that binds and removes anionic molecules, obtained from PallCorporation, Ann Arbor, Mich., USA). This membrane was first washed with4 mL of 1 M NaOH, 4 mL of 1 M NaCl and 10 mL of 100 mM citrate buffer(pH 6) in turn, and liposomes were warmed for 10 min at 37° C. beforebeing filtered. Secondly, the hollow fiber filtration membrane wasPolysulfone (part number P/N: X1AB-100-20P).

The Z average particle diameter, polydispersity index and encapsulationefficiency of the liposomes were as follows:

RV Zav (nm) pdI % encapsulation Preparation RV01 (10) 158.6 0.088 90.7(A) RV01 (08) 156.8 0.144 88.6 (A) RV01 (05) 136.5 0.136 99 (B) RV01(09) 153.2 0.067 76.7 (A) RV05 (01) 148 0.127 80.6 (A) RV05 (02) 177.20.136 72.4 (A) RV01 (10) 134.7 0.147 87.8 * (A) RV13 (02) 128.3 0.179 97(A) * For this RV01(10) formulation the nucleic acid was DNA not RNA

Serum was collected for antibody analysis on days 14, 36 and 49. Spleenswere harvested from mice at day 49 for T cell analysis.

F-specific serum IgG titers (GMT) were as follows:

RV Day 14 Day 36 Naked DNA plasmid 439 6712 Naked A317 RNA 78 2291 RV01(10) 3020 26170 RV01 (08) 2326 9720 RV01 (05) 5352 54907 RV01 (09) 442851316 RV05 (01) 1356 5346 RV05 (02) 961 6915 RV01 (10) DNA 5 13 RV13(02) 644 3616

The proportion of T cells which are cytokine-positive and specific forRSV F51-66 peptide are as follows, showing only figures which arestatistically significantly above zero:

CD4+CD8− CD4−CD8+ RV IFNγ IL2 IL5 TNFα IFNγ IL2 IL5 TNFα Naked DNAplasmid 0.04 0.07 0.10 0.57 0.29 0.66 Naked A317 RNA 0.04 0.05 0.08 0.570.23 0.67 RV01 (10) 0.07 0.10 0.13 1.30 0.59 1.32 RV01 (08) 0.02 0.040.06 0.46 0.30 0.51 RV01 (05) 0.08 0.12 0.15 1.90 0.68 1.94 RV01 (09)0.06 0.08 0.09 1.62 0.67 1.71 RV05 (01) 0.06 0.04 0.19 RV05 (02) 0.050.07 0.11 0.64 0.35 0.69 RV01 (10) DNA 0.03 0.08 RV13 (02) 0.03 0.040.06 1.15 0.41 1.18

Thus the liposome formulations significantly enhanced immunogenicityrelative to the naked RNA controls, as determined by increasedF-specific IgG titers and T cell frequencies. Plasmid DNA formulatedwith liposomes, or delivered naked using electroporation, wassignificantly less immunogenic than liposome-formulated self-replicatingRNA.

The RV01 and RV05 RNA vaccines were more immunogenic than the RV13(DOTAP) vaccine. These formulations had comparable physicalcharacteristics and were formulated with the same self-replicating RNA,but they contain different cationic lipids. RVO1 and RVO5 both have atertiary amine in the headgroup with a pKa of about 5.8, and alsoinclude unsaturated alkyl tails. RV13 has unsaturated alkyl tails butits headgroup has a quaternary amine and is very strongly cationic.These results suggest that lipids with tertiary amines with pKas in therange 5.0 to 7.6 are superior to lipids such as DOTAP, which arestrongly cationic, when used in a liposome delivery system for RNA.

Further Alternatives to DLinDMA

The cationic lipid in RVO1 liposomes (DLinDMA) was replaced by RV16,RV17, RV18 or RV19. Total IgG titers are shown in FIG. 17. The lowestresults are seen with RV19 i.e. the DOTMA quaternary amine.

BHK Expression

Liposomes with different lipids were incubated with BHK cells overnightand assessed for protein expression potency. From a baseline with RVO5lipid expression could be increased 18× by adding 10%1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPyPE) to theliposome, 10× by adding 10% 18:2 (cis) phosphatidylcholine, and 900× byinstead using RV01.

RSV Immunogenicity in Different Mouse Strains

Replicon “vA142” encodes the full-length wild type surface fusion (F)glycoprotein of RSV but with the fusion peptide deleted, and the 3′ endis formed by ribozyme-mediated cleavage. It was tested in threedifferent mouse strains.

BALB/c mice were given bilateral intramuscular vaccinations (50 μL perleg) on days 0 and 22. Animals were divided into 8 test groups (5animals per group) and a naïve control (2 animals):

-   -   Group 1 were given naked replicon (1 μg).    -   Group 2 were given 1μg replicon delivered in liposomes        “RV01(37)” with 40% DlinDMA, 10% DSPC, 48% Chol, 2%        PEG-conjugated DMG.    -   Group 3 were given the same as group 2, but at 0.1 μg RNA.    -   Group 4 were given 1 μg replicon in “RV17(10)” liposomes (40%        RV17 (see above), 10% DSPC, 49.5% cholesterol, 0.5% PEG-DMG).    -   Group 5 were 1 μg replicon in “RV05(11)” liposomes (40% RV07        lipid, 30% 18:2 PE (DLoPE, 28% cholesterol, 2% PEG-DMG).    -   Group 6 were given 0.1 μg replicon in “RV17(10)” liposomes.    -   Group 7 were given 5 μg RSV-F subunit protein adjuvanted with        aluminium hydroxide.    -   Group 8 were a naïve control (2 animals)

Sera were collected for antibody analysis on days 14, 35 and 49.F-specific serum IgG GMTs were:

Day 1 2 3 4 5 6 7 8 14 82 2463 1789 2496 1171 1295 1293 5 35 1538 3418125605 23579 13718 8887 73809 5

At day 35 F-specific IgG1 and IgG2a titers (GMT) were as follows:

IgG 1 2 3 4 5 6 7 IgG1 94 6238 4836 7425 8288 1817 78604 IgG2a 538677064 59084 33749 14437 17624 24

RSV serum neutralizing antibody titers at days 35 and 49 were as follows(data are 60% plaque reduction neutralization titers of pools of 2-5mice, 1 pool per group):

Day 1 2 3 4 5 6 7 8 35 <20 143 20 101 32 30 111 <20 49 <20 139 <20 83 4132 1009 <20

Spleens were harvested at day 49 for T cell analysis. Average netF-specific cytokine-positive T cell frequencies (CD4+ or CD8+) were asfollows, showing only figures which were statistically significantlyabove zero (specific for RSV peptides F51-66, F164-178, F309-323 forCD4+, or for peptides F85-93 and F249-258 for CD8+):

CD4+CD8− CD4−CD8+ Group IFNγ IL2 IL5 TNFα IFNγ IL2 IL5 TNFα 1 0.03 0.060.08 0.47 0.29 0.48 2 0.05 0.10 0.08 1.35 0.52 1.11 3 0.03 0.07 0.060.64 0.31 0.61 4 0.05 0.09 0.07 1.17 0.65 1.09 5 0.03 0.08 0.07 0.650.28 0.58 6 0.05 0.07 0.07 0.74 0.36 0.66 7 0.02 0.04 0.04 8

C57BL/6 mice were immunised in the same way, but a 9th group receivedVRPs (1×10⁶ IU) expressing the full-length wild-type surface fusionglycoprotein of RSV (fusion peptide deletion).

Sera were collected for antibody analysis on days 14, 35 & 49.F-specific IgG titers (GMT) were:

Day 1 2 3 4 5 6 7 8 9 14 1140 2133 1026 2792 3045 1330 2975 5 1101 351721 5532 3184 3882 9525 2409 39251 5 12139

At day 35 F-specific IgG1 and IgG2a titers (GMT) were as follows:

IgG 1 2 3 4 5 6 7 8 IgG1 66 247 14 328 468 92 56258 79 IgG2a 2170 76855055 6161 1573 2944 35 14229

RSV serum neutralizing antibody titers at days 35 and 49 were as follows(data are 60% plaque reduction neutralization titers of pools of 2-5mice, 1 pool per group):

Day 1 2 3 4 5 6 7 8 9 35 <20 27 29 22 36 <20 28 <20 <20 49 <20 44 30 2336 <20 33 <20 37

Spleens were harvested at day 49 for T cell analysis. Average netF-specific cytokine-positive T cell frequencies (CD8+) were as follows,showing only figures which were statistically significantly above zero(specific for RSV peptides F85-93 and F249-258):

CD4−CD8+ Group IFNγ IL2 IL5 TNFα 1 0.42 0.13 0.37 2 1.21 0.37 1.02 31.01 0.26 0.77 4 1.26 0.23 0.93 5 2.13 0.70 1.77 6 0.59 0.19 0.49 7 0.100.05 8 9 2.83 0.72 2.26

Nine groups of C3H/HeN mice were immunised in the same way. F-specificIgG titers (GMT) were:

Day 1 2 3 4 5 6 7 8 9 14 5 2049 1666 1102 298 984 3519 5 806 35 15227754 19008 17693 3424 6100 62297 5 17249

At day 35 F-specific IgG1 and IgG2a titers (GMT) were as follows:

IgG 1 2 3 4 5 6 7 8 IgG1 5 1323 170 211 136 34 83114 189 IgG2a 302136941 78424 67385 15667 27085 3800 72727

RSV serum neutralizing antibody titers at days 35 and 49 were asfollows:

Day 1 2 3 4 5 6 7 8 9 35 <20 539 260 65 101 95 443 <20 595 49 <20 456296 35 82 125 1148 <20 387

Thus three different lipids (RV01, RV05, RV17; pKa 5.8, 5.85, 6.1) weretested in three different inbred mouse strains. For all 3 strains RV01was more effective than RV17; for BALB/c and C3H strains R05 was lesseffective than either RV01 or RV17, but it was more effective in B6strain. In all cases, however, the liposomes were more effective thantwo cationic nanoemulsions which were tested in parallel.

CMV Immunogenicity

RV01 liposomes with DLinDMA as the cationic lipid were used to deliverRNA replicons encoding cytomegalovirus (CMV) glycoproteins. The “vA160”replicon encodes full-length glycoproteins H and L (gH/gL), whereas the“vA322” replicon encodes a soluble form (gHsol/gL). The two proteins areunder the control of separate subgenomic promoters in a single replicon;co-administration of two separate vectors, one encoding gH and oneencoding gL, did not give good results.

BALB/c mice, 10 per group, were given bilateral intramuscularvaccinations (50 μL per leg) on days 0, 21 and 42 with VRPs expressinggH/gL (1×10⁶ IU), VRPs expressing gHsol/gL (1×10⁶ IU) and PBS as thecontrols. Two test groups received lug of the vA160 or vA322 repliconformulated in liposomes (40% DlinDMA, 10% DSPC, 48% Chol, 2% PEG-DMG;made using method (A) as discussed above, but with 150 μg RNA batchsize).

The vA160 liposomes had a Zav diameter of 168nm, a pdI of 0.144, and87.4% encapsulation. The vA322 liposomes had a Zav diameter of 162 nm, apdI of 0.131, and 90% encapsulation.

The replicons were able to express two proteins from a single vector.

Sera were collected for immunological analysis on day 63 (3wp3). CMVneutralization titers (the reciprocal of the serum dilution producing a50% reduction in number of positive virus foci per well, relative tocontrols) were as follows:

gH/gL VRP gHsol/gL VRP gH/gL liposome gHsol/gL liposome 4576 2393 424010062

RNA expressing either a full-length or a soluble form of the CMV gH/gLcomplex thus elicited high titers of neutralizing antibodies, as assayedon epithelial cells. The average titers elicited by theliposome-encapsulated RNAs were at least as high as for thecorresponding VRPs.

It will be understood that the invention has been described by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

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[45] WO2011/057020.

1. A method of eliciting an immune response against an immunogen in amammal, the method comprising administering to the mammal an effectiveamount of a formulation to elicit the immune response, the formulationcomprising: ribonucleic acid (RNA) molecules comprising a sequence thatencodes the immunogen; and lipid nanoparticles (LNPs) comprising lipidscomprising first lipids, second lipids, polyethylene glycol-conjugated(PEG-conjugated) lipids, and cholesterol; wherein: at least half of theRNA molecules are comprised within the LNPs; the second lipids compriseanionic lipids or neutral zwitterionic lipids; the first lipids comprisea tertiary amine and have a pKa from 5.0 to 7.6; at least half of thefirst lipids are neutrally charged when the first lipids are at a pHthat is above the pKa; and at least half of the first lipids arepositively charged when the first lipids are at a pH that is below thepKa; and whereby the pKa is defined by the following: (1) admixing thefirst lipids with ethanol and fluorescent probe6-(p-Toluidino)-2-naphthalenesulfonic acid (TNS), thereby obtaining alipid/TNS mixture; (2) separately admixing each of a plurality of asodium salt buffer with a portion of the lipid/TNS mixture, wherein thesodium salt buffer comprises 20 mM sodium phosphate, 25 mM sodiumcitrate, 20 mM sodium acetate, and 150 mM sodium chloride, wherein eachof the plurality of the sodium salt buffer has a different pH and theplurality of the sodium salt buffer has a range of pH from 4.4 to 11.12,thereby obtaining a plurality of pH-varied lipid/TNS mixtures; (3)measuring the absolute fluorescence at a wavelength of 431 nm with anexcitation wavelength of 322 nm and a cut-off below a wavelength of 420nm of each of the plurality of the pH-varied lipid/TNS mixtures, therebyobtaining an absolute fluorescence for each of the plurality of thepH-varied lipid/TNS mixtures; (4) measuring the absolute fluorescence ata wavelength of 431 nm with an excitation wavelength of 322 nm and acut-off below a wavelength of 420 nm of an empty vessel used in themeasuring of (3), thereby obtaining a blank fluorescence; (5)subtracting the blank fluorescence from each of the absolutefluorescences of the plurality of the pH-varied lipid/TNS mixtures,thereby obtaining a blank-subtracted fluorescence for each of theplurality of the pH-varied lipid/TNS mixtures; (6) normalizing each ofthe blank-subtracted fluorescences of the plurality of the pH-variedlipid/TNS mixtures to the blank-subtracted fluorescence of the pH-variedlipid/TNS mixture that was obtained from the admixing in (2) with thesodium salt buffer that had the lowest pH of the first sodium saltbuffers, thereby obtaining a relative fluorescence for each of theplurality of the pH-varied lipid/TNS mixtures, the relative fluorescencebeing 1 for the pH-varied lipid/TNS mixture that was obtained from theadmixing in (2) with the sodium salt buffer that had the lowest pH ofthe first sodium salt buffers; (7) obtaining a line of best fit of thepHs of the sodium salt buffers versus the respective relativefluorescences of the plurality of pH-varied lipid/TNS mixtures; and (8)defining the pKa as the pH on the line of best fit at which a relativefluorescence of 0.5 is obtained.
 2. The method of claim 1, wherein theimmunogen comprises two or more different immunogens.
 3. The method ofclaim 1, wherein the immunogen comprises a viral immunogen, a bacterialimmunogen, a fungal immunogen, or a parasitic immunogen.
 4. The methodof claim 3, wherein the viral immunogen comprises a hepadnavirusimmunogen and the immune response is at least against hepadnavirus. 5.The method of claim 3, wherein the viral immunogen comprises aherpesvirus immunogen and immune response is at least againstherpesvirus.
 6. The method of claim 3, wherein the viral immunogencomprises a papillomavirus immunogen and the immune response is at leastagainst papillomavirus.
 7. The method of claim 3, wherein the viralimmunogen comprises a coronavirus immunogen and the immune response isat least against coronavirus.
 8. The method of claim 3, wherein theviral immunogen comprises a cytomegalovirus immunogen and the immuneresponse is at least against cytomegalovirus.
 9. The method of claim 3,wherein the viral immunogen comprises an Epstein-Barr virus immunogenand the immune response is at least against Epstein-Barr virus.
 10. Themethod of claim 3, wherein the bacterial immunogen comprises aHelicobacter pylori immunogen and the immune response is at leastagainst Helicobacter pylori.
 11. The method of claim 3, wherein thefungal immunogen comprises a Malassezia spp. immunogen and the immuneresponse is at least against Malassezia spp.
 12. The method of claim 1,wherein the immunogen comprises a tumor antigen and the immune responseis at least against a tumor expressing the tumor antigen.
 13. The methodof claim 1, wherein the immunogen comprises a viral immunogen, andwherein the viral immunogen comprises a polyomavirus immunogen, anoncovirus immunogen, a lentivirus immunogen, a flavivirus immunogen, anorthomyxovirus immunogen, a paramyxovirus immunogen, or a picornavirusimmunogen.
 14. The method of claim 1, wherein from 0.5 ml to 1.0 ml ofthe formulation is administered.
 15. The method of claim 1, wherein themethod comprises administering multiple unit doses of the formulationfour weeks apart to the mammal.
 16. The method of claim 1, furthercomprising administering a booster dose of the effective amount of theformulation to the mammal.
 17. The method of claim 16, whereinadministering the booster dose to the mammal is at least 6 months afteradministering the effective amount of the formulation.
 18. The method ofclaim 1, wherein the immune response comprises an antibody response. 19.The method of claim 18, wherein the antibody response comprises aneutralizing antibody response.
 20. The method of claim 1, wherein theLNPs comprise from 35 mol % to 50 mol % of the cholesterol.
 21. Themethod of claim 1, wherein the LNPs comprise from 1 mol % to 6 mol % ofthe PEG-conjugated lipids.
 22. The method of claim 1, wherein the immuneresponse comprises a CD8+ T cell response.
 23. The method of claim 22,wherein the CD8+ T cell response results in production of cytokines inthe mammal.
 24. The method of claim 23, wherein the cytokines compriseIFNγ, TNF-α, or IL-2.
 25. The method of claim 1, wherein thePEG-conjugated lipids comprise a PEG that has a molecular weight of 2000Daltons.
 26. The method of claim 1, wherein the PEG-conjugated lipidscomprise 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol. 27.The method of claim 1, wherein the neutral zwitterionic lipids comprise1,2-distearoyl-sn-glycero-3-phosphocholine or dimyristoylphosphatidylethanolamine.
 28. The method of claim 1, wherein the RNAmolecules further comprise a 7′-methylguanosine, a tri-phosphate bridge,and a 5′ first ribonucleoside, and wherein the 7′-methylguanosine islinked 5′-to-5′ to the 5′ first ribonucleoside by the triphosphatebridge.
 29. The method of claim 1, wherein the pKa is from 5.6 to 6.8.30. The method of claim 25, wherein the PEG-conjugated lipids comprise1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol.